Thermoreversible compositions for administration of therapeutic agents

ABSTRACT

The present invention relates to compositions and methods of inducing the self-renewal of stem/progenitor supporting cells, including inducing the stem/progenitor cells to proliferate while maintaining, in the daughter cells, the capacity to differentiate into hair cells.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisional application No. 62/302,747, filed Mar. 2, 2016, the entire contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to thermoreversible compositions of poloxamers and methods of using same for administration of therapeutic agents

BACKGROUND OF THE INVENTION

Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are often considered “functional excipients” because they are essential components and play an important role in a formulation.

Poloxamers are synthetic triblock co-polymers with the following general structure formula:

wherein a=2-130 and b=15-67

Non-limiting examples of poloxamers include: poloxamer 124, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. The chemical characteristics of each poloxamer are determined by the chain length of the polyethylene (EO) units and the polyoxypropylene (PO) units, molecular weight and degree of hydrophobicity/hydrophilicity. Some poloxamers are liquid at room temperature (124) whereas others are a solid (184, 185, 188, 237, 338, and 407). Poloxamers have a variety of uses including as emulsifying agents, solubilizing agents and stabilizing agents. For example, poloxamer 188 has been used as an emulsifying agent for fluorocarbons used as artificial blood substitutes and in the preparation of solid-dispersions. They are also used as wetting agents in ointments, suppository bases, and gels, as well as tablet binders and coatings. In some cases, poloxamers have been used for solubilizing formulations containing otic therapeutic agents and their delivering. In some cases, a poloxamer formulation is created containing active agents that is liquid when at 4° C. which gels when heated at or above room temperature. This enables the formulation to be injected through a small needle and gel when it hits the middle ear. In these cases the amounts of poloxamer used for solubilization was about 15-17 wt %. At concentrations above 15-17 wt %, poloxamers will typically form gels that are not flowable even with cooled to 4° C., making them unsuitable for injection. There is a need for injectable formulations of therapeutic agents containing a poloxamer with gelation temperatures of at least 10 degrees or at least body temperature, where the poloxamer is at concentrations higher than about 15-17 wt %.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a pharmaceutically active therapeutic agent or a pharmaceutically acceptable salt thereof;

b) a poloxamer at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the poloxamer comprises at least one of Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407. In some embodiments, the poloxamer comprises a mixture of two or more of Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407. In some embodiments, the mixture of two or more poloxamers comprises Poloxamer 407 and Poloxamer 124. In another embodiment, the Poloxamer comprises at least one of Poloxamer 188 and Poloxamer 407 or mixtures thereof. In some embodiments, the poloxamer is Poloxamer 407.

In some embodiments, the poloxamer can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C.

In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide. In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid. In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition). In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

In some embodiments of the poloxamer containing pharmaceutical composition, the pharmaceutically active therapeutic agent is selected from the group consisting of a wnt agonist, a GSK3 inhibitor, a 2-pyrimidinylaminoethylamino-2-pyridine containing compound, a N-(1H-pyrazol-4-yl)-nicotinamide containing compound, and a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione containing compound.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a wnt agonist or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a GSK3 inhibitor or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising

a) a 2-pyrimidinylaminoethylamino-2-pyridine compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

A further aspect of the present disclosure provides poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamide containing compound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,]indol-7-yl)-1H-pyrrole-2,5-dione containing compound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a compound of Formula I:

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the amount of Valproic acid in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition). In some embodiments, the composition comprises at least 4 wt % Valproic acid. In some embodiments, the composition comprises at least 8 wt % Valproic acid. In other embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprising Formula I, comprises between about 2 wt % Valproic acid and about 20 wt %. In other embodiments, the composition comprising Formula I, comprises about 2 wt % Valproic acid. In other embodiments, the composition comprising Formula I, comprises about 4 wt % Valproic acid. In other embodiments, the composition comprising Formula I, comprises about 8 wt % Valproic acid. In other embodiments, the composition comprising Formula I, comprises about 12 wt % Valproic acid. In further embodiments, the composition comprising Formula I, comprises about 16 wt % Valproic acid. In further embodiments, the composition comprising Formula I, comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a compound of Formula II

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the amount of Valproic acid in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprising the compound of Formula II, comprises at least 4 wt % Valproic acid. In some embodiments, the composition comprising the compound of Formula II, comprises at least 8 wt % Valproic acid. In other embodiments, the composition comprising the compound of Formula II, comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprising the compound of Formula II, comprises at least 16 wt % Valproic acid. In other embodiments, the composition comprising the compound of Formula II, comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprising Formula II, comprises between about 2 wt % Valproic acid and about 20 wt %. In other embodiments, the composition comprising Formula II, comprises about 2 wt % Valproic acid. In other embodiments, the composition comprising Formula II, comprises about 4 wt % Valproic acid. In other embodiments, the composition comprising Formula II, comprises about 8 wt % Valproic acid. In other embodiments, the composition comprising Formula II, comprises about 12 wt % Valproic acid. In further embodiments, the composition comprising Formula II, comprises about 16 wt % Valproic acid. In further embodiments, the composition comprising Formula II, comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a compound of Formula III

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the amount of Valproic acid in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %.

In some embodiments, the composition comprising the compound of Formula III, comprises at least 4 wt % Valproic acid. In some embodiments, the composition comprising the compound of Formula III, comprises at least 8 wt % Valproic acid. In other embodiments, the composition comprising the compound of Formula III, comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprising the compound of Formula III, comprises at least 16 wt % Valproic acid. In other embodiments, the composition comprising the compound of Formula III, comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprising Formula III, comprises between about 2 wt % Valproic acid and about 20 wt %. In other embodiments, the composition comprising Formula III, comprises about 2 wt % Valproic acid. In other embodiments, the composition comprising Formula III, comprises about 4 wt % Valproic acid. In other embodiments, the composition comprising Formula III, comprises about 8 wt % Valproic acid. In other embodiments, the composition comprising Formula III, comprises about 12 wt % Valproic acid. In further embodiments, the composition comprising Formula III, comprises about 16 wt % Valproic acid. In further embodiments, the composition comprising Formula III, comprises about 20 wt % Valproic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the solubility of Poloxamer 407 (P407) at 16.7% of w/w (16.7 grams Poloxamer 407 to 83.3 grams of a solution that contains % 1 Molar valproic acid sodium salt/5% weight DMSO and water), 28.6% w/w, 33.3% w/w, 37.5% w/w, 41.2% w/w, and 50.0% w/w in 1M Valproic acid sodium salt and 5% dimethylsulfoxide (DMSO) in water. Percent w/w of Poloxamer 407 left to right as shown: 16.7%, 28.6%, 33.3%, 37.5%, 41.2%, and 50.0%.

FIG. 2 is a photograph showing the solubility of Poloxamer 407 (P407) at 20% w/w, 23.1% w/w, and 25.9% w/w Poloxamer 407 in 1M Valproic acid sodium salt and 5% dimethylsulfoxide (DMSO). Percent w/w of P407 left to right as shown: 20% w/w, 23.1% w/w, and 25.9% w/w.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides poloxamer containing pharmaceutical compositions which exhibit a gelation temperature greater than 10° C. These compositions comprise pharmaceutically active therapeutic agents or their pharmaceutically acceptable salts. Moreover, these pharmaceutical compositions comprise both poloxamer at greater than or equal to 21 wt % of the pharmaceutical composition and a carboxylic acid containing compound and have a gelation temperature of greater than 10° C. The carboxylic acid containing compound is in an amount of between at least 2 wt % and 20 wt %. These compositions are described herein as well as some non-limiting uses of same.

Definitions

In this application, the use of “or” means “and/or” unless stated otherwise. As used in this application, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

“Administration” refers to introducing a substance into a subject. In some embodiments, administration is auricular, intraauricular, intracochlear, intravestibular, or transtympanically, e.g., by injection. In some embodiments, administration is directly to the inner ear, e.g., injection through the round or oval window, otic capsule, or vestibular canals. In some embodiments, administration is directly into the inner ear via a cochlear implant delivery system. In some embodiments, the substance is injected transtympanically to the middle ear. In certain embodiments “causing to be administered” refers to administration of a second component after a first component has already been administered (e.g., at a different time and/or by a different actor).

An “antibody” refers to an immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.

As used herein, an “agonist” is an agent that causes an increase in the expression or activity of a target gene, protein, or a pathway, respectively. Therefore, an agonist can bind to and activate its cognate receptor in some fashion, which directly or indirectly brings about this physiological effect on the target gene or protein. An agonist can also increase the activity of a pathway through modulating the activity of pathway components, for example, through inhibiting the activity of negative regulators of a pathway. Therefore, a “Wnt agonist” can be defined as an agent that increases the activity of Wnt pathway, which can be measured by increased TCF/LEF-mediated transcription in a cell. Therefore, a “Wnt agonist” can be a true Wnt agonist that binds and activates a Frizzled receptor family member, including any and all of the Wnt family proteins, an inhibitor of intracellular beta-catenin degradation, and activators of TCF/LEF.

An “antagonist” refers to an agent that binds to a receptor, and which in turn decreases or eliminates binding by other molecules.

“Anti-sense” refers to a nucleic acid sequence, regardless of length, that is complementary to the coding strand or mRNA of a nucleic acid sequence. Antisense RNA can be introduced to an individual cell, tissue or organanoid. An anti-sense nucleic acid can contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.

As referred to herein, a “complementary nucleic acid sequence” is a nucleic acid sequence capable of hybridizing with another nucleic acid sequence comprised of complementary nucleotide base pairs. By “hybridize” is meant pair to form a double-stranded molecule between complementary nucleotide bases (e.g., adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA) under suitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

“Auricular administration” refers to a method of using a catheter or wick device to administer a composition across the tympanic membrane to the inner ear of the subject. To facilitate insertion of the wick or catheter, the tympanic membrane may be pierced using a suitably sized syringe or pipette. The devices could also be inserted using any other methods known to those of skill in the art, e.g., surgical implantation of the device. In particular embodiments, the wick or catheter device may be a stand-alone device, meaning that it is inserted into the ear of the subject and then the composition is controllably released to the inner ear. In other particular embodiments, the wick or catheter device may be attached or coupled to a pump or other device that allows for the administration of additional compositions. The pump may be automatically programmed to deliver dosage units or may be controlled by the subject or medical professional.

“Biocompatible Matrix” as used herein is a polymeric carrier that is acceptable for administration to humans for the release of therapeutic agents. A Biocompatible Matrix may be a biocompatible gel or foam.

“Cell Aggregate” as used herein shall mean a body cells in the Organ of Corti that have proliferated to form a cluster of a given cell type that is greater than 40 microns in diameter and/or produced a morphology in which greater than 3 cell layers reside perpendicular to the basilar membrane. A “Cell Aggregate” can also refer a process in which cell division creates a body of cells that cause one or more cell types to breach the reticular lamina, or the boundary between endolymph and perilymph

“Cell Density” as used herein in connection with a specific cell type is the mean number of that cell type per area in a Representative Microscopy Sample. The cell types may include but are not limited to Lgr5⁺ cells, hair cells, or supporting cells. The Cell Density may be assessed with a given cell type in a given organ or tissue, including but not limited to the cochlea or Organ of Corti. For instance, the Lgr5⁺ Cell Density in the Organ of Corti is the Cell Density of Lgr5⁺ cells as measured across the Organ of Corti. Typically, supporting cells and Lgr5⁺ cells will be enumerated by taking cross sections of the Organ of Corti. Typically, hair cells will be enumerated by looking down at the surface of the Organ of Corti, though cross sections may be used in some instances, as described in a Representative Microscopy Sample. Typically, Cell Density of Lgr5⁺ cells will be measured by analyzing whole mount preparations of the Organ of Corti and counting the number of Lgr5 cells across a given distance along the surface of the epithelia, as described in a Representative Microscopy Sample. Hair cells may be identified by their morphological features such as bundles or hair cell specific stains (e.g., Myosin VIIa, Prestin, vGlut3, Pou4f3, Espin, conjugated-Phalloidin, PMCA2, Ribeye, Atoh1, etc). Lgr5+ cells may be identified by specific stains or antibodies (e.g., Lgr5-GFP transgenic reporter, anti-Lgr5 antibody, etc.)

“Cochlear Concentration” as used herein will be the concentration of a given agent as measured through sampling cochlear fluid. Unless otherwise noted, the sample should contain a substantial enough portion of the cochlear fluid so that it is approximately representative of the average concentration of the agent in the cochlea. For example, samples may be drawn from a vestibular canal, and a series of fluid samples drawn in series such that individual samples are comprised of cochlear fluid in specified portions of the cochlea

“Complementary nucleic acid sequence” refers to a nucleic acid sequence capable of hybridizing with another nucleic acid sequence comprised of complementary nucleotide base pairs.

“Cross-Sectional Cell Density” as used herein in connection with a specific cell type is the mean number of that cell type per area of cross section through a tissue in a Representative Microscopy Sample. Cross sections of the Organ of Corti can also be used to determine the number of cells in a given plane. Typically, hair cells Cross-sectional Cell Density will be measured by analyzing whole mount preparations of the Organ of Corti and counting the number of hair cells across a given distance in cross sections taken along a portion of the epithelia, as described in a Representative Microscopy Sample. Typically, Cross-sectional Cell Density of Lgr5⁺ cells will be measured by analyzing whole mount preparations of the Organ of Corti and counting the number of Lgr5⁺ cells across a given distance in cross sections taken along a portion of the epithelia, as described in a Representative Microscopy Sample. Hair cells may be identified by their morphological features such as bundles or hair cell specific stains (suitable stains include e.g., Myosin VIIa, Prestin, vGlut3, Pou4f3, conjugated-Phalloidin, PMCA2, Atoh1, etc.). Lgr5⁺ cells may be identified by specific stains or antibodies (suitable stains and antibodies include fluorescence in situ hybridization of Lgr5 mRNA, Lgr5-GFP transgenic reporter system, anti-Lgr5 antibodies, etc.).

“Decreasing” refers to decreasing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, for example, as compared to the level of reference.

“Decreases” also means decreases by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, for example, as compared to the level of a reference.

“Differentiation Period” as used herein is the duration of time in which there is an Effective Stemness Driver Concentration

“Effective Concentration” may be the Effective Stemness Driver Concentration for a Stemness Driver.

“Effective Release Rate” (mass/time) as used herein is the Effective Concentration (mass/volume)*30 uL/1 hour.

“Effective Stemness Driver Concentration” is the minimum concentration of a Stemness Driver that induces at least 1.5-fold increase in number of LGR5+ cells in a Stem Cell Proliferation Assay compared to the number of Lgr5⁺ cells in a Stem Cell Proliferation Assay performed without the Stemness Driver and with all other components present at the same concentrations.

“Eliminate” means to decrease to a level that is undetectable.

“Engraft” or “engraftment” refers to the process of stem or progenitor cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue. “Epithelial progenitor cell” refers to a multipotent cell which has the potential to become restricted to cell lineages resulting in epithelial cells.

“Epithelial stem cell” refers to a multipotent cell which has the potential to become committed to multiple cell lineages, including cell lineages resulting in epithelial cells.

“Fragment” refers to a portion of a polypeptide or nucleic acid molecule. This portion contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

“Gelation Temperature,” as used herein is defined as meaning the temperature below which a composition is in liquid form and above which the composition forms a semi-solid, for example, a gel.

“GSK3beta,” “GSK3β,” and “GSK3B” as used interchangeably herein are acronyms for glycogen synthase kinase 3 beta,

“GSK3 inhibitor” is a composition that inhibits the activity of any GSK3.

“GSK3beta inhibitor” is a composition that inhibits the activity of GSK3beta.

“GSK3alpha,” “GSK3α,” and “GSK3A” as used interchangeably herein are acronyms for glycogen synthase kinase 3 alpha,

“GSK3alpha inhibitor” is a composition that inhibits the activity of GSK3alpha.

“Hybridize” refers to pairing to form a double-stranded molecule between complementary nucleotide bases (e.g., adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA) under suitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

An “inhibitor” refers to an agent that causes a decrease in the expression or activity of a target gene or protein, respectively. An “antagonist” can be an inhibitor, but is more specifically an agent that binds to a receptor, and which in turn decreases or eliminates binding by other molecules.

As used herein, an “inhibitory nucleic acid” is a double-stranded RNA, RNA interference, miRNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. Typically, expression of a target gene is reduced by 10%, 25%, 50%, 75%, or even 90-100%.

“In vitro Lgr5 activity” refers to the level of expression or activity of Lgr5 in an in vitro population of cells. It may be measured, for example, in cells derived from a Lgr5-GFP expressing mouse such as a B6.129P2-Lgr5tm1(cre/ERT2)Cle/J mouse (also known as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No: 008875) by dissociating cells to single cells, staining with propidium iodide (PI), and analyzing the cells using a flow cytometer for Lgr5-GFP expression. Inner ear epithelial cells from wild-type (non-Lgr5-GFP) mice that passing the same culturing and analyzing procedures can be used as a negative control. Typically, two populations of cells are shown in the bivariate plot with GFP/FITC as one variable, which include both GFP positive and GFP negative populations. Lgr5-positive cells are identified by gating GFP positive cell population. The percentage of Lgr5-positive cells is measured by gating GFP positive cell population against both GFP negative population and the negative control. The number of Lgr5-positive cells is calculated by multiplying the total number of cells by the percentage of Lgr5-positive cells. For cells derived from non-Lgr5-GFP mice, Lgr5 activity can be measured using an anti-Lgr5 antibody or quantitative-PCR on the Lgr5 gene.

“In vivo Lgr5 activity” as used herein is the level of expression or activity of Lgr5 in a subject. It may be measured, for example, by removing an animal's inner ear and measuring Lgr5 protein or Lgr5 mRNA. Lgr5 protein production can be measured using an anti-Lgr5 antibody to measure fluorescence intensity as determined by imaging cochlear samples, where fluorescence intensity is used as a measure of Lgr5 presence. Western blots can be used with an anti-Lgr5 antibody, where cells can be harvested from the treated organ to determine increases in Lgr5 protein. Quantitative-PCR or RNA in situ hybridization can be used to measure relative changes in Lgr5 mRNA production, where cells can be harvested from the inner ear to determine changes in Lgr5 mRNA. Alternatively, Lgr5 expression can be measured using an Lgr5 promoter driven GFP reporter transgenic system, where the presence or intensity GFP fluoresce can be directly detected using flow cytometry, imaging, or indirectly using an anti-GFP antibody.

“Increases” also means increases by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, for example, as compared to the level of a as compared to the level of a reference standard.

“Increasing” refers to increasing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100% or more, for example, as compared to the level of a reference.

“Intraauricular administration” refers to administration of a composition to the middle or inner ear of a subject by directly injecting the composition.

“Intracochlear” administration refers to direct injection of a composition across the tympanic membrane and across the round or oval window membrane into the cochlea.

“Intravestibular” administration refers to direct injection of a composition across the tympanic membrane and across the round or oval window membrane into the vestibular organs.

“Isolated” refers to a material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings.

“Lgr5” is an acronym for the Leucine-rich repeat-containing G-protein coupled receptor 5, also known as G-protein coupled receptor 49 (GPR49) or G-protein coupled receptor 67 (GPR67). It is a protein that in humans is encoded by the Lgr5 gene.

“Lgr5 activity” is defined as the level of activity of Lgr5 in a population of cells. In an in vitro cell population, Lgr5 activity may be measured in an in vitro Lgr5 Activity assay. In an in vivo cell population, Lgr5 activity may be measured in an in vivo Lgr5 Activity assay.

“Lgr5⁺ cell” or “Lgr5-positive cell” as used herein is a cell that expresses Lgr5. “Lgr5⁻ cell” as used herein is a cell that is not Lgr5⁺.

“Lineage Tracing” as used herein is using a mouse line that enables fate tracing of any cell that expresses a target gene at the time of reporter induction. This can include hair cell or supporting cells genes (Sox2, Lgr5, MyosinVIIa, Pou4f3, etc). For example, lineage tracing may use an Lgr5-EGFP-IRES-creERT2 mouse crossed with a reporter mouse, which upon induction, allows one to trace the fate of cells that expressed Lgr5 at the time of induction. By further example, Lgr5 cells can be isolated into single cells and cultured in a Stem Cell Proliferation Assay to generate colonies, then subsequently differentiated in a Differentiation Assay and analyzed for cell fate by staining for hair cell and/or supporting cell proteins and determining the reporter colocalization with either hair cell or supporting cell staining to determine the Lgr5 cells' fate. In addition, lineage tracing can be performed in cochlear explants to track supporting cell or hair cell fate within the intact organ after treatment. For example, Lgr5 cell fate can be determined by isolating the cochlea from a Lgr5-EGFP-IRES-creERT2 mouse crossed with a reporter mouse, and inducing the reporter in Lgr5 cells before or during treatment. The organ can then be analyzed for cell fate by staining for hair cell and/or supporting cell proteins and determining the reporter colocalization with either hair cell or supporting cell staining to determine the Lgr5 cells' fate. In addition, lineage tracing can be performed in vivo track supporting cell or hair cell fate within the intact organ after treatment. For example, Lgr5 cell fate can be determined inducing a reporter in an Lgr5-EGFP-IRES-creERT2 mouse crossed with a reporter mouse, treating the animal, then isolating the cochlea. The organ can then be analyzed for cell fate by staining for hair cell and/or supporting cell proteins and determining the reporter colocalization with either hair cell or supporting cell staining to determine the Lgr5 cells' fate. Lineage tracing may be performed using alternative reporters of interest as is standard in the art.

“Mammal” refers to any mammal including but not limited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

“Mean Release Time” as used herein is the time in which one-half of an agent is released into phosphate buffered saline from a carrier in a Release Assay.

“Native Morphology” as used herein is means that tissue organization largely reflects the organization in a healthy tissue.

“Non-human mammal”, as used herein, refers to any mammal that is not a human.

As used in relevant context herein, the term “number” of cells can be 0, 1, or more cells.

“Organ of Corti” as used herein refers to the sensory cells (inner and outer hair cells) of the hearing organ located in the cochlea.

“Organoid” or “epithelial organoid” refers to a cell cluster or aggregate that resembles an organ, or part of an organ, and possesses cell types relevant to that particular organ.

“Population” of cells refers to any number of cells greater than 1, but is at least 1×10³ cells, at least 1×10⁴ cells, at least at least 1×10⁵ cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹ cells, or at least 1×10¹⁰ cells.

“Progenitor cell” as used herein refers to a cell that, like a stem cell, has the tendency to differentiate into a specific type of cell, but is already more specific than a stem cell and is pushed to differentiate into its “target” cell.

“Proliferation Period” as used herein is the duration of time in which there is an Effective Stemness Driver Concentration.

“Reference” means a standard or control condition (e.g., untreated with a test agent or combination of test agents).

“Release Assay” as used herein is a test in which the rate of release of an agent from a Biocompatible Matrix through dialysis membrane to a saline environment. An exemplary Release Assay may be performed by placing 30 microliters of a composition in 1 ml Phosphate Buffered Saline inside saline dialysis bag with a suitable cutoff, and placing the dialysis bag within 10 mL of Phosphate Buffered Saline at 37 C. The dialysis membrane size may be chosen based on agent size in order to allow the agent being assessed to exit the membrane. For small molecule release, a 3.5-5 kDa cutoff may be used. The Release Rate for a composition may change over time and may be measured in 1 hour increments.

“Representative Microscopy Sample” as used herein describes a sufficient number of fields of view within a cell culture system, a portion of extracted tissue, or an entire extracted organ that the average feature size or number being measured can reasonably be said to represent the average feature size or number if all relevant fields were measured. For example, in order to assess the hair cell counts at a frequency range on the Organ of Corti, ImageJ software (NIH) can used to measure the total length of cochlear whole mounts and the length of individual counted segments. The total number of inner hair cells, outer hair cells, and supporting cells can be counted in the entire or fraction of any of the four cochlear segments of 1200-1400 μm (apical, mid-apical, mid-basal, and basal) at least 3 fields of view at 100 μm field size would be reasonably considered a Representative Microscopy Sample. A Representative Microscopy sample can include measurements within a field of view, which can be measured as cells per a given distance. A Representative Microscopy sample can be used to assess morphology, such as cell-cell contacts, cochlear architecture, and cellular components (e.g., bundles, synapses).

“Rosette Patterning” is a characteristic cell arrangement in the cochlea in which <5% hair cells are adjacent to other hair cells.

The term “sample” refers to a volume or mass obtained, provided, and/or subjected to analysis. In some embodiments, a sample is or comprises a tissue sample, cell sample, a fluid sample, and the like. In some embodiments, a sample is taken from (or is) a subject (e.g., a human or animal subject). In some embodiments, a tissue sample is or comprises brain, hair (including roots), buccal swabs, blood, saliva, semen, muscle, or from any internal organs, or cancer, precancerous, or tumor cells associated with any one of these. A fluid may be, but is not limited to, urine, blood, ascites, pleural fluid, spinal fluid, and the like. A body tissue can include, but is not limited to, brain, skin, muscle, endometrial, uterine, and cervical tissue or cancer, precancerous, or tumor cells associated with any one of these. In an embodiment, a body tissue is brain tissue or a brain tumor or cancer. Those of ordinary skill in the art will appreciate that, in some embodiments, a “sample” is a “primary sample” in that it is obtained from a source (e.g., a subject); in some embodiments, a “sample” is the result of processing of a primary sample, for example to remove certain potentially contaminating components and/or to isolate or purify certain components of interest.

“Self-renewal” refers to the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter cells with development potentials that are indistinguishable from those of the mother cell. Self-renewal involves both proliferation and the maintenance of an undifferentiated state.

“siRNA” refers to a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3′ end. These dsRNAs can be introduced to an individual cell or culture system. Such siRNAs are used to downregulate mRNA levels or promoter activity.

“Stem cell” refers to a multipotent cell having the capacity to self-renew and to differentiate into multiple cell lineages.

“Stem Cell Differentiation Assay” as used herein is an assay to determine the differentiation capacity of stem cells. In an exemplary Stem Cell Differentiation Assay, the number of cells for an initial cell population is harvested from a Atoh1-GFP mouse between the age of 3 to 7 days, by isolating the Organ of Corti sensory epithelium, dissociating the epithelium into single cells, and passing the cells through a 40 um cell strainer. Approximately 5000 cells are entrapped in 40 μl of culture substrate (for example: Matrigel (Corning, Growth Factor Reduced)) and placed at the center of wells in a 24-well plate with 500 μl of an appropriate culture media, growth factors and agent being tested. Appropriate culture media and growth factors include Advanced DMEM/F12 with media Supplements (1×N2, 1×B27, 2 mM Glutamax, 10 mM HEPES, 1 mM N-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin) and growth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50 ng/ml IGF-1) as well as the agent(s) being assessed are added into each well. Cells are cultured for 10 days in a standard cell culture incubator at 37° C. and 5% CO₂, with media change every 2 days. These cells are then cultured by removing the Stem Cell Proliferation Assay agents and replacing with Basal culture media and molecules to drive differentiation. An appropriate Basal culture media is Advanced DMEM/F12 supplemented with 1×N2, 1×B27, 2 mM Glutamax, 10 mM HEPES, 1 mM N-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin and appropriate molecules to drive differentiation are 3 μM CHIR99021 and 5 μM DAPT for 10 days, with media change every 2 days. The number of hair cells in a population may be measured by using flow cytometry for GFP. Hair cell differentiation level can further be assessed using qPCR to measure hair cell marker (e.g., Myo7a) expression level normalized using suitable and unregulated references or housekeeping genes (e.g., Hprt). Hair cell differentiation level can also be assessed by immunostaining for hair cell markers (e.g., Myosin7a, vGlut3, Espin, PMCAs, Ribeye, conjugated-phalloidin, Atoh1, Pou4f3, etc). Hair cell differentiation level can also be assessed by Western Blot for Myosin7a, vGlut3, Espin, PMCAs, Prestin, Ribeye, Atoh1, and Pou4f3.

“Stem Cell Assay” as used herein is an assay in which a cell or a cell population are tested for a series of criteria to determine whether the cell or cell population are stem cells or enriched in stem cells or stem cell markers. In a stem cell assay, the cell/cell populations are tested for stem cell characteristics such as expression of Stem Cell Markers, and further optionally are tested for stem cell function, including the capacity of self-renewal and differentiation.

“Stem Cell Proliferator” as used herein is a composition that induces an increase in a population of cells which have the capacity for self-renewal and differentiation.

“Stem Cell Proliferation Assay” as used herein is an assay to determine the capacity for agent(s) to induce the creation of stem cells from a starting cell population. In an exemplary Stem Cell Proliferation Assay, the number of cells for an initial cell population is harvested from a Lgr5-GFP mouse such as a B6.129P2-Lgr5tm1(cre/ERT2)Cle/J mouse (also known as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No: 008875) between the age of 3 to 7 days, by isolating the Organ of Corti sensory epithelium and dissociating the epithelium into single cells. Approximately 5000 cells are entrapped in 40 μl of culture substrate (for example: Matrigel (Corning, Growth Factor Reduced)) and placed at the center of wells in a 24-well plate with 500 μl of an appropriate culture media, growth factors and agent being tested. Appropriate culture media and growth factors include Advanced DMEM/F12 with media Supplements (1×N2, 1×B27, 2 mM Glutamax, 10 mM HEPES, 1 mM N-acetylcysteine, and 100 Um′ Penicillin/100 μg/ml Streptomycin) and growth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50 ng/ml IGF-1) as well as the agent(s) being assessed are added into each well. Cells are cultured for 10 days in a standard cell culture incubator at 37° C. and 5% CO₂, with media change every 2 days. The number of Lgr5⁺ cells is quantified by counting the number of cells identified as Lgr5+ in an In vitro Lgr5 activity assay. The fraction of cells that are Lgr5⁺ is quantified by dividing the number of cells identified as Lgr5⁺ in a cell population by the total number of cells present in the cell population. The average Lgr5⁺ activity of a population is quantified by measuring the average mRNA expression level of Lgr5 of the population normalized using suitable and unregulated references or housekeeping genes (e.g., Hprt). The number of hair cells in a population may be measured by staining with hair cell marker (e.g., Myosin VIIa), or using an endogenous reporter of hair cell genes (e.g., Pou4f3-GFP, Atoh1-nGFP) and analyzing using flow cytometry. The fraction of cells that are hair cells is quantified by dividing the number of cells identified as hair cells in a cell population by the total number of cells present in the cell population. Lgr5 activity can be measured by qPCR.

“Stem Cell Markers” as used herein can be defined as gene products (e.g., protein, RNA, etc) that specifically expressed in stem cells. One type of stem cell marker is gene products that are directly and specifically support the maintenance of stem cell identity. Examples include Lgr5 and Sox2. Additional stem cell markers can be identified using assays that have been described in the literature. To determine whether a gene is required for maintenance of stem cell identity, gain-of-function and loss-of-function studies can be used. In gain-of-function studies, over expression of specific gene product (the stem cell marker) would help maintain the stem cell identity. While in loss-of-function studies, removal of the stem cell marker would cause loss of the stem cell identity or induced the differentiation of stem cells. Another type of stem cell marker is gene that only expressed in stem cells but does not necessary to have specific function to maintain the identity of stem cells. This type of markers can be identified by comparing the gene expression signature of sorted stem cells and non-stem cells by assays such as micro-array and qPCR. This type of stem cell marker can be found in the literature. (e.g., Liu Q. et al., Int J Biochem Cell Biol. 2015 March; 60:99-111. http://www.ncbi.nlm.nih.gov/pubmed/25582750). Potential stem cell markers include Ccdc121, Gdf10, Opcm1, Phex, etc. The expression of stem cell markers such as Lgr5 or Sox2 in a given cell or cell population can be measure using assays such as qPCR, immunohistochemistry, western blot, and RNA hybridization. The expression of stem cell markers can also be measured using transgenic cells express reporters which can indicate the expression of the given stem cell markers, e.g., Lgr5-GFP or Sox2-GFP. Flow cytometry analysis can then be used to measure the activity of reporter expression. Fluorescence microscopy can also be used to directly visualize the expression of reporters. The expression of stem cell markers may further be determined using microarray analysis for global gene expression profile analysis. The gene expression profile of a given cell population or purified cell population can be compared with the gene expression profile of the stem cell to determine similarity between the 2 cell populations. Stem cell function can be measured by colony forming assay or sphere forming assay, self-renewal assay and differentiation assay. In colony (or sphere) forming assay, when cultured in appropriate culture media, the stem cell should be able to form colonies, on cell culture surface (e.g., cell culture dish) or embedded in cell culture substrate (e.g., Matrigel) or be able to form spheres when cultured in suspension. In colony/sphere forming assay, single stem cells are seeded at low cell density in appropriate culture media and allowed to proliferate for a given period of time (7-10 days). Colony formed are then counted and scored for stem cell marker expression as an indicator of stemness of the original cell. Optionally, the colonies that formed are then picked and passaged to test its self-renewal and differentiation potential. In self-renewal assay, when cultured in appropriate culture media, the cells should maintain stem cell marker (e.g., Lgr5) expression over at least one (e.g., 1, 2, 3, 4, 5, 10, 20, etc) cell divisions. In a Stem Cell Differentiation Assay, when cultured in appropriate differentiation media, the cells should be able to generate hair cell which can be identified by hair cell marker expression measured by qPCR, immunostaining, western blot, RNA hybridization or flow cytometry.

“Stemness Driver” as used herein is a composition that induces proliferation of LGR5⁺ cells, upregulates Lgr5 in cells, or maintains Lgr5 expression in cells, while maintaining the potential for self-renewal and the potential to differentiate into hair cells. Generally, stemness drivers upregulate at least one biomarker of post-natal stem cells. Stemness Drivers include but are not limited to Wnt agonists and GSK3Beta inhibitors.

“Subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

“Supporting Cell” as used herein in connection with a cochlear epithelium comprises epithelial cells within the organ of Corti that are not hair cells. This includes inner pillar cells, outer pillar cells, inner phalangeal cells, Deiter cells, Hensen cells, Boettcher cells, and/or Claudius cells.

“Synergy” or “synergistic effect” is an effect which is greater than the sum of each of the effects taken separately; a greater than additive effect.

“TgfBeta inhibitor” as used herein is a composition that reduces activity of TgfBeta

“Thermoreversible gel” means a material that reversibly changes from a liquid to a gel with a change in temperature.

“Tissue” is an ensemble of similar cells from the same origin that together carries out a specific function including, for example, tissue of cochlear, such as the Organ of Corti.

“Transtympanic” administration refers to direct injection of a composition across the tympanic membrane into the middle ear.

“Treating” as used herein in connection with a cell population means delivering a substance to the population to effect an outcome. In the case of in vitro populations, the substance may be directly (or even indirectly) delivered to the population. In the case of in vivo populations, the substance may be delivered by administration to the host subject.

“Valproic acid” (VPA) has chemical formula C₈H₁₆O₂ and the following alternate name: 2-propylpentanoic acid. Its chemical structure is as follows:

As used herein, the term “Valproic acid” also includes derivatives of Valproic Acid including its salts, amides, prodrugs and esters.

“Wnt activation” as used herein in connection with a composition is an activation of the Wnt signaling pathway.

The use of “or” means “and/or” unless stated otherwise. As used in this application, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical compositions.

“Pharmaceutically acceptable salt” includes both acid and base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid,/toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. For example, inorganic salts include, but are not limited to, ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Non-limiting examples of organic bases used in certain embodiments include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

“w/w” refers to the ratio of reagents in a composition measured using the weight of the reagent in grams, milligrams, or kilograms.

“wt %’ refers to the percent of that agent in the composition measured using the weight of the agent measured in grams, milligrams, or kilograms.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

One aspect the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a pharmaceutically active therapeutic agent or a pharmaceutically acceptable salt thereof;

b) a poloxamer at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition to have a gelation temperature of greater than 10° C.

In some embodiments, the poloxamer comprises at least one of Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407. In some embodiments, the poloxamer comprises mixtures of two or more of Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407. In some embodiments, the mixture of two or more poloxamers comprises Poloxamer 407 and Poloxamer 124. In another embodiment, the Poloxamer comprises at least one of Poloxamer 188 and Poloxamer 407 or mixtures thereof. In some embodiments, the poloxamer is Poloxamer 407.

In some embodiments, the poloxamer can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide.

In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid. In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

In some embodiments of the poloxamer containing pharmaceutical composition, the pharmaceutically active therapeutic agent is selected from the group consisting of a wnt agonist, a GSK3 inhibitor, a 2-pyrimidinylaminoethylamino-2-pyridine containing compound, a N-(1H-pyrazol-4-yl)-nicotinamide containing compound, and a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione containing compound.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising

a) a wnt agonist or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the wnt agonist is selected from the group consisting of

WNT Agonists CAS Number Compound 1 1084833-94-2 Compound 25 1084834-05-8 BML-284 853220-52-7 IQ 1 331001-62-8 DCA 56-47-3 QS 11 944328-88-5 WASP-1, 352328-82-6 ZINC00087877 WAY 316606 915759-45-4 WAY-262611 1123231-07-1 HLY78 854847-61-3 SKL2001 909089-13-0

In other embodiments, the wnt agonist is selected from the group consisting of the Compound 1, Compound 25, BML-284, QS-11, WAY 316606, WAY-262611, SKL2001, and ISX-9.

In some embodiments, Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide. In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid.

In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a GSK3 inhibitor or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the pharmaceutically active therapeutic agent is a GSK3 inhibitor. In some embodiments, the GSK3 inhibitor is a GSK3 alpha inhibitor. In some embodiments, the GSK alpha inhibitor is selected from the group consisting of

GSKalpha inhibitor CAS Number GSK-3 inhibitor XXII 1195901-31-5 AT 7519 844442-38-2 Compound 4a 1627557-91-8 Compound 4t 1627558-10-4 Compound 4z 1627558-16-0 Compound 14 583038-63-5 Compound 23 583038-76-0 Pyrazolopyridine 34 583039-27-4 Compound 18 405223-20-3 Compound 19 405223-71-4 Compound 15b 1374671-66-5 Compound 14d 1374671-64-3 Compound 27 1820758-44-8 AZD1080 612487-72-6 Compound 39 1772824-10-8 Compound 29 1772823-37-6 Compound 33 1772823-64-9 Tivantinib 905854-02-6 I5 264217-24-5 Compound 90 91322-11-1 Compound 92 1043429-30-6 Compound A-OS1 1291104-51-2 1292843-11-8 Compound 3 1498285-39-4 1498285-48-5 Compound (R)-DW12 1047684-07-0 BRD4003 chiral 1597439-60-5 BRD4003 chiral 1597439-59-2 Compound 8 1597439-01-4 Compound 9 1597439-02-5 2056261-29-9 Compound 11 1597439-12-7 BRD1172 1597438-86-2 Compound 16 1597440-17-9 BRD1652 1597438-93-1 AR-A014418 487021-52-3 CREB knockdown

In some embodiments, the GSK3-alpha inhibitors are selected from the group consisting of GSK-alpha Inhibitor XXII, Compound 4a, Compound 4t, Compound 4z, Compound 14, Compound 23, Compound 18, Compound 19, Compound 15b, Compound 14d, AZD-1080, Compound 39, Compound 29, Compound 33, Compound 92, and Compound Λ-OS1

In some embodiments, the pharmaceutically active therapeutic agent is a GSK3 inhibitor is a GSK3 beta inhibitor.

In some embodiments, the GSK3 Inhibitor is selected from the group consisting of:

GSK3β Inhibitor CAS Reg. No. Valproic Acid, Sodium 99-66-1 Salt Bikinin 188011-69-0 Hymenialdisine 82005-12-7 Aloisine A 496864-16-5 Aloisine B 496864-14-3 TWS119 1507095-58-0 CT20026 403808-63-9 CHIR99021 (CT99021) 252917-06-9 CHIR98014 (CT98014) 252935-94-7 CHIR98023 (CT98023) 252904-84-0 CHIR98024 (CT98024) 556813-39-9 GSK-3β Inhibitor XVIII 1139875-74-3 CGP60474 164658-13-3 AZD2858 (AR28) 486424-20-8 CID 755673 521937-07-5 TCS 2002 1005201-24-0 Dibromocantharelline 101481-34-9 ML320 1597438-84-0 Flavopiridol 146426-40-6 Compound 100 744255-19-4 Hymenidin 107019-95-4 6-Bromoindirubin-3- 667463-85-6 acetoxime GSK-3 Inhibitor IX 667463-62-9 Indirubin-3′-monoxime 160807-49-8 5-Iodo-indirubin-3′- 331467-03-9 monoxime Indirubin-5-sulfonic acid 331467-05-1 sodium salt Indirubin 479-41-4 GSK-3 Inhibitor X 740841-15-0 Lithium Chloride Beryllium Zinc Tungstate Compound 39 1772824-10-8 Compound 29 1772823-37-6 Compound 33 1772823-64-9 Compound 29 436866-61-4 Compound 46 682807-74-5 Compound 5a 436866-54-5 GF109203x 176504-36-2 Ro318220 125314-64-9 Bisindolylmaleimide X 131848-97-0 HCl Enzastaurin (LY317615) 170364-57-5 I5 264217-24-5 SB-216763 280744-09-4 SB-415286 (SB-41528) 264218-23-7 3F8 159109-11-2 TCS 21311 1260181-14-3 GSK-3 inhibitor 1 603272-51-1 LY2090314 603288-22-8 603281-31-8 603281-31-8 IM-12 1129669-05-1 Compound 34 396091-16-0 KT 5720 108068-98-0 Isogranulatimide 244148-46-7 GSK-3β Inhibitor XI 626604-39-5 BIP-135 941575-71-9 CP21R7 125314-13-8 Tivantinib 905854-02-6 Compound A-OS1 1291104-51-2, 1292843-11-8 HB12 800384-87-6 DW12 861251-33-4 NP309 937810-13-4 (RRu)-HB1229 (RRu)-NP549 Compound 3 1498285-39-4, 1498285-48-5 Compound (R)-DW12 1047684-07-0 Staurosporine 62996-74-1 GSK-3beta Inhibitor XXVI 871843-09-3 Manzamine A 104196-68-1 TC-G 24 1257256-44-2 Compound 14d 1374671-64-3 Compound 15b 1374671-66-5 Compound 20x 1005201-80-8 GSK-3 Inhibitor II 478482-75-6 GSK3 Inhibitor, 2 1377154-01-2 SU9516 77090-84-1 AZD-1080 612487-72-6 Kenpaullone 142273-20-9 Compound 17b 408532-42-3 Azakenpaullone 676596-65-9 Alsterpaullone 237430-03-4 Alsterpaullone CN Ethyl 852529-97-0 Cazpaullone 914088-64-5 FRATtide L803 L803-mts GSK-3 Inhibitor XXII 1195901-31-5 Compound 4a 1627557-91-8 Compound 4t 1627558-10-4 Compound 4z 1627558-16-0 AT 7519 844442-38-2 Pyrazolopyridine 9 923029-74-7 Pyrazolopyridine 18 405221-39-8 Pyrazolopyridine 34 583039-27-4 Compound 14 583038-63-5 Compound 23 583038-76-0 Compound 14 583038-63-5 Compound 18 405223-20-3 Compound 19 405223-71-4 NSC 693868 (Compound 40254-90-8 1) Compound 150 1282042-18-5 GSK-3 Inhibitor XIII 404828-08-6 VP0.7 331963-23-6 1132813-46-7 1132812-98-6 950727-66-9 NSC 693868 (Compound 40254-90-8 1) Compound 17 62673-69-2 GSK-3β Inhibitor VII 99-73-0 GSK-3β Inhibitor VI 62673-69-2 Palinurin 254901-27-4 Tricantin 853885-55-9 GSK-3β Inhibitor I 327036-89-5 NP031115 1400575-57-6 NP031112 (Tideglusib) 865854-05-3 Compound 90 91322-11-1 Compound 92 1043429-30-6 GSK-3β Inh. VIII AR- 487021-52-3 A014418 A-1070722 1384424-80-9 NP-103 No Structure CG-301338 No Structure SAR 502250 No Structure XD-4241 No Structure CEP-16805 No Structure AZ13282107 No Structure SAR 502250 (Sanofi) 1073653-58-3 Compound 27 2025388-25-2 Compound 12 2025388-10-5

In certain embodiments, the GSK3β Inhibitor are selected from the group consisting of Valproic Acid Sodium Salt, CT20026, CHIR99021 (CT99021), CHIR98014 (CT98014), CHIR98023 (CT98023), CHIR98024 (CT98024), TCS 2002, Compound 39, Compound 29, Compound 33, TCS 21311, LY2090314, 603281-31-8, Compound 34, Compound 14d, Compound 15b, Compound 20x, AZD-1080, Kenpaullone, Cazpaullone, GSK-3 Inhibitor XXII, Compound 4a, Compound 4t, Compound 4z, Pyrazolopyridine 9, Compound 14, Compound 23, Compound 14, Compound 18, and Compound 19.

In certain embodiments, the GSK3β Inhibitor are selected from the group consisting of Valproic Acid Sodium Salt, CHIR99021 (CT99021), CHIR98014 (CT98014), CHIR98023 (CT98023), CHIR98024 (CT98024), Compound 39, Compound 29, LY2090314, 603281-31-8, Compound 34, Compound 14d, Compound 15b, Compound 20x, AZD-1080, Cazpaullone, GSK-3 Inhibitor XXII, Compound 4t, Compound 4z, Pyrazolopyridine 9, Compound 14, Compound 23, Compound 14, Compound 18, and Compound 19.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide. In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid. In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a 2-pyrimidinylaminoethylamino-2-pyridine compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the 2-pyrimidinylaminoethylamino-2-pyridine compound is a 2,4-dichlorophenyl-5-(1H-imidazol-2-yl)-2-pyrimidinylaminoethylamino-3-pyridine compound. In some embodiments, the pyrimidine containing compound is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile which has the Formula I and pharmaceutically acceptable salts thereof:

The compound of Formula I is also known as CHIR99021.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide. In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid. In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamide containing compound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containing compound is an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamide containing compound. In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containing compound is N-(3-((3-isopropoxypropyl)carbamoyl)-1H-pyrazol-4-yl)-6-methylnicotinamide which has the Formula II and pharmaceutically acceptable salts thereof:

The compound of Formula II is also known as GSK-3 Inhibitor XXII.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide. In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid.

In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) a carboxylic acid containing compound;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is a 3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione containing compound. In some embodiments, the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is a 3-(imidazo[1,2-a]pyridin-3-yl)-4-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione containing compound. In some embodiments, 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is 3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione which has the Formula III and pharmaceutically acceptable salts thereof:

The compound of Formula III is also known as LY2090314.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In any of the foregoing embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is a substituted C₅-C₉ straight or branched chain alkyl-CO₂H, wherein the substituent is —NH₂. In some examples the carboxylic acid containing compound is an amino substituted 2-propylpentanoic acid. In some embodiments, the amino substituted 2-propylpentanoic acid is selected from the group consisting of 5-amino-2-propylpentanoic acid, 4-amino-2-propylpentanoic acid, 3-amino-2-propylpentanoic acid, and 2-amino-2-propylpentanoic acid.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₆-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₈-C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the carboxylic acid containing compound is an unsubstituted C₉ branched straight chain alkyl-CO₂H.

In some embodiments, the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid.

In some embodiments, the Valproic acid can be in the form of an amide. In some embodiments, the amide form of Valproic acid is the condensation product with an amino acid. In some embodiments, the amide of Valproic acid is selected from the group consisting of

In any of the foregoing embodiments, the amount of the carboxylic acid containing compound in the pharmaceutical composition is between at least 2 wt % (weight carboxylic acid containing compound/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % carboxylic acid. In other embodiments, the composition comprises at least 8 wt % carboxylic acid. In some embodiments, the composition comprises at least 12 wt % carboxylic acid. In some embodiments, the composition comprises at least 16 wt % carboxylic acid. In still other embodiments, the composition comprises at least 20 wt % carboxylic acid.

In other embodiments, the composition comprises about 4 wt % carboxylic acid. In other embodiments, the composition comprises about 8 wt % carboxylic acid. In other embodiments, the composition comprises about 12 wt % carboxylic acid. In further embodiments, the composition comprises about 16 wt % carboxylic acid. In further embodiments, the composition comprises about 20 wt % carboxylic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a wnt agonist or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a GSK3 inhibitor or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a 2-pyrimidinylaminoethylamino-2-pyridine compound;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the 2-pyrimidinylaminoethylamino-2-pyridine compound is a 2,4-dichlorophenyl-5-(1H-imidazol-2-yl)-2-pyrimidinylaminoethylamino-2-pyridine compound. In some embodiments, the pyrimidine containing compound is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile which has the Formula I and pharmaceutically acceptable salts thereof:

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

-   -   a) an N-(1H-pyrazol-4-yl)-nicotinamide containing compound or a         pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containing compound is an N-(alkylcarbamoyl)-1H-pyrazol-4-yl)-nicotinamide containing compound.

In some embodiments, the N-(1H-pyrazol-4-yl)-nicotinamide containing compound is N-(3-((3-isopropoxypropyl)carbamoyl)-1H-pyrazol-4-yl)-6-methylnicotinamide which has the structure in Formula II and pharmaceutically acceptable salts thereof:

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compound containing compound or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compound is a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione containing compound. In some embodiments, the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compound is a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione containing compound. In some embodiments, the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl) containing compound is 3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione which has the Formula III and pharmaceutically acceptable salts thereof:

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising

a) a compound of the Formula I

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising:

a) a compound of Formula II

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

A further aspect of the present disclosure provides a poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising

a) a compound of Formula III

or a pharmaceutically acceptable salt thereof;

b) Poloxamer 407 in weight percentage of more than 21% wt; and

c) Valproic acid;

wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.

In some embodiments, the Poloxamer 407 can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.

In some embodiments, the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between is between about 15° C. and about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is between about 24° C. and about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 15° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 20° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 24° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 26° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 28° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 30° C. In some embodiments, the gelation temperature of the pharmaceutical composition is about 32° C.

In any of the foregoing embodiments, the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %. In some embodiments, the composition comprises at least 4 wt % Valproic acid. In other embodiments, the composition comprises at least 8 wt % Valproic acid. In some embodiments, the composition comprises at least 12 wt % Valproic acid. In some embodiments, the composition comprises at least 16 wt % Valproic acid. In still other embodiments, the composition comprises at least 20 wt % Valproic acid.

In other embodiments, the composition comprises about 4 wt % Valproic acid. In other embodiments, the composition comprises about 8 wt % Valproic acid. In other embodiments, the composition comprises about 12 wt % Valproic acid. In further embodiments, the composition comprises about 16 wt % Valproic acid. In further embodiments, the composition comprises about 20 wt % Valproic acid.

In any of the foregoing aspects and embodiments, the poloxamer can be in a concentration between 21 wt % and 40 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 21 wt % and 30 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 23 wt % and 29 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration between 23 wt % and 27 wt % relative to the composition. In another embodiment, the poloxamer is in a concentration of 25 wt % relative to the composition.

Poloxamers are commercially available (e.g., Pluronic® polyols are available from BASF Corporation). Alternatively, poloxamers can be synthesized by known techniques.

In one or more embodiments the viscosity of the composition at about body temperature is substantially higher than the viscosity of the composition at 4° C. In some embodiments the viscosity of the composition at 4° C. is between 100 cp and 100,000 cp. In some embodiments the viscosity of the composition at 4° C. is between 100 cp and 10,000 cp. In some embodiments the viscosity of the composition at 4° C. is between 100 cp and 1,000 cp. In some embodiments the viscosity of the composition at 37° C. is between 50,000 cp and 500,000 cp. In some embodiments the viscosity of the composition at 37° C. is between 100,000 cp and 300,000 cp. In some embodiments the viscosity of the composition at 37° C. is at least 10-fold greater than the viscosity of the composition at 4° C. In some embodiments the viscosity of the composition at 37° C. is at least 50-fold greater than the viscosity of the composition at 4° C.

In other embodiments, the carboxylic acid is in a concentration between 20 and 160 mg/mL relative to the composition. In other embodiments, the carboxylic acid is in a concentration of approximately 87.5 mg/mL relative to the composition.

In some embodiments, it is desirable that the formulation be sufficiently viscous that it remain in an area of administration for a suitable period of time, e.g., 1 min, 5 min, 10 min, 30 min., 1 hour, 5 hours, 12, hours, 1 day, 2 days, 7 days. The desired area may be external to the eardrum, but in contact therewith, e.g., where it can be desired that a layer of solution remain in contact with the eardrum (e.g., not flow out of ear) when the recipient subject is upright; even if some of a viscous formulation exits the ear canal, a viscous film of layer can, in some embodiments, be let in contact with the eardrum.

In some embodiments, the final formulation is a liquid at storage temperature (4° C.), and formed a semisolid gel above its liquid-gel transition temperature (37° C.). In other embodiments, compositions have a viscosity of less than 100,000 centipoise (cps) at 25° C. Compositions also have a minimum yield stress that is sufficient for maintaining the formulation against the tympanic membrane. Yield stress is the amount of force that causes a solid material to exhibit liquid-like behavior in that it continues to deform with no further increase in stress. Minimum yield stress is dependent on the thickness of the applied gel, but is independent of the geometry of the gel and the temperature of the environment. Minimum yield stress of a composition refers to an applied gel having a thickness of 4 mm and a density of 1 g/L. Yield stress (σ₀) is represented as σ₀=μgh, where ρ is density, g is the acceleration due to gravity, and h is the layer thickness. Typically, minimum yield stress is about 39 pascals (Pa).

Viscogenic agents are typically polymers or other chemical moieties that increase the viscosity of a fluid. Suitable viscogenic agents, when included in a composition, allow the composition to transform from a liquid-like state (e.g., flowable) at 25° C. to a solid-like state (e.g., a gel) after contact with the tympanic membrane, and can be non-biodegradable, (e.g., not broken down by chemicals or enzymes naturally present in a mammal, or biodegradable). Compositions include an amount of viscogenic agent effective to yield a viscosity of the composition of less than 100,000 cps at 25° C. (e.g., less than 90,000, 60,000, 30,000, 20,000, or 10,000 cps) and, generally, a minimum yield stress of 39 Pa after application to the tympanic membrane. Typically, a composition includes 0.05 to 50% of a viscogenic agent (e.g., 0.15 to 25, 5 to 45, 10 to 40, 12 to 37, 15 to 35, 17 to 33, or 20 to 30% of a viscogenic agent).

Exemplary viscogenic agents include gellan (GELRITE or KELCOGEL), CARBOPOL 940 with hydroxypropylmethylcellulose (HPMC), N-isopropyl acrylamide (NiPAAm) with sodium acrylate and n-N-alkylacrylamide, polyacrylic acid with polyethylene glycol (PEG) or polymethacryhc acid with PEG, cellulose acetate hydrogen phthalate latex (CAP), sodium alginate, and nonionic surfactants such as poloxamers (PLURIONIC) and polyoxamine (TETRONIC) reversible temperature-dependent gelling systems.

Among other things, the compositions provided herein are useful for the prophylaxis and/or treatment of acute and chronic ear disease and hearing loss, dizziness and balance problems especially of sudden hearing loss, acoustic trauma, hearing loss due to chronic noise exposure, presbycusis, trauma during implantation of the inner ear prosthesis (insertion trauma), dizziness due to diseases of the inner ear area, dizziness related and/or as a symptom of Meniere's disease, vertigo related and/or as a symptom of Meniere's disease, tinnitus, and hearing loss due to antibiotics and cytostatics and other drugs.

Stem cells exhibit an extraordinary ability to generate multiple cell types in the body. Besides embryonic stem cells, tissue specific stem cells serve a critical role during development as well as in homeostasis and injury repair in the adult. Stem cells renew themselves through proliferation as well as generate tissue specific cell types through differentiation. The characteristics of different stem cells vary from tissue to tissue, and are determined by their intrinsic genetic and epigenetic status. However, the balance between self-renewal and differentiation of different stem cells are all stringently controlled. Uncontrolled self-renewal may lead to overgrowth of stem cells and possibly tumor formation, while uncontrolled differentiation may exhaust the stem cell pool, leading to an impaired ability to sustain tissue homeostasis. Thus, stem cells continuously sense their environment and appropriately respond with proliferation, differentiation or apoptosis. It would be desirable to drive regeneration by controlling the timing and extent of stem cell proliferation and differentiation. Controlling the proliferation with small molecules that are cleared over time would allow for control of the timing and extent of stem cell proliferation and differentiation. Remarkably, tissue stem cells from different tissues share a limited number of signaling pathways for the regulation of their self-renewal and differentiation, albeit in a very context dependent manner. Some of these pathways are the Wnt and GSK3-beta pathways.

Lgr5 is expressed across a diverse range of tissues and has been identified as a biomarker of adult stem cells in a variety of tissues such as the gut epithelia (Barker et al. 2007), kidney, hair follicle, and stomach (Barker et al, 2010; Haegebarth & Clevers, 2009). For example, it was first published in 2011, that mammalian inner ear hair cells are derived from LGR5⁺ cells (Chai et al, 2011, Shi et al. 2012). Lgr5 is a known component of the Wnt/beta-catenin pathway, which has been shown to play major roles in differentiation, proliferation, and inducing stem cell characteristics (Barker et al. 2007).

Permanent damage to the hair cells of the inner ear results in sensorineural hearing loss, leading to communication difficulties in a large percentage of the population. Hair cells are the receptor cells that transduce the acoustic stimulus. Regeneration of damaged hair cells would provide an avenue for the treatment of a condition that currently has no therapies other than prosthetic devices. Although hair cells do not regenerate in the mammalian cochlea, new hair cells in lower vertebrates are generated from epithelial cells, called supporting cells, that surround hair cells.

Prior work has focused on transdifferentiation of supporting cells into hair cells through activation or forced expression of genes that lead to hair cell formation, with a particular focus on mechanisms to enhance expression of Atoh1 (Bermingham et al., 1999; Zheng and Gao, 2000; Izumikawa et al., 2005; Mizutari et al., 2013). Interestingly, cells transduced with Atoh1 vectors have been shown to acquire vestibular phenotypes (Kawamoto et al., 2003; Huang et al., 2009; Yang et al., 2012, 2013), and lack complete development. As mentioned, upregulating Atoh1 via gene insertion has been shown to create non-cochlear cell types that behave in a manner that is not found within the native cochlea. In addition, these methods increase hair cell numbers but decrease supporting cell numbers. Since supporting cells are known to have specialized roles (Ramirez-Camancho 2006, Dale and Jagger 2010), loss of these cells could create problems in proper cochlear function.

In one aspect the present disclosure provides compositions useful for controlled proliferation of stem cells comprising administering to a cell population an effective amount of a composition herein described.

Among the various aspects of the present disclosure, therefore, may be noted a method for activating the Wnt pathway in a cell population to increase the capacity of the population for self-renewal, i.e., the capacity for repeated generation of daughter cells with equivalent proliferation and ‘cell fate specification’ potential, and differentiation, i.e., the capacity for generation of daughter cells specified for differentiation. In one embodiment, the cell population is a cochlear supporting cell population. The Wnt pathway may also be activated upstream of the c-myc gene in members of the population and without any genetic modification of the population. Further, the Wnt pathway can also be activated by small molecules that transiently induce such activity. Additionally, the supporting cell population includes supporting cells that are LGR5⁺ and endogenous to the Organ of Corti.

In certain embodiments a composition includes a Notch agonist or an HDAC inhibitor selected such as Valproic acid or a prodrug, ester, salt form, or amide thereof. Optionally, the composition also includes a Stemness Driver. Optionally, the Stemness Driver is a Wnt agonist. In some embodiments, the Wnt agonist is a GSK3 inhibitor. In some embodiments the Wnt agonist is CHIR99021, LY2090314, AZD 1080, or GSK3 Inhibitor XXII.

In certain embodiments, therefore, the present disclosure provides compositions that have the capacity to induce self-renewal of a population of supporting cells. These compositions have the capacity to activate pathways and mechanisms that are known to be involved in inducing stem cell properties, such as those used to create “induced pluripotent stem cells”. These pathways are activated with small molecules. For example, a composition when applied in vitro to a supporting cell population may induce the population to proliferate to a high degree and in high purity in a Stem Cell Proliferation Assay, and also allow the population to differentiate into a high purity population of hair cells in a Stem Cell Differentiation Assay. In one such embodiment, the composition induces and maintains stem cell properties by proliferating to produce stem cell that can divide for many generations and maintain the ability to have a high proportion of the resulting cells differentiate into hair cells. Further, the proliferating stem cells express stem cell markers which may include one or more of Lgr5, Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1, Tc11, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1, Smad2, smad2/3, smad4, smad5, and smad7.

In certain embodiments, the disclosure provides a method of and compositions for generating hair cells, the method comprising: administering or causing to be administered to a stem cell population (e.g., of an in vitro, ex vivo, or in vivo sample/subject) a composition comprising a GSK3-beta inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and/or Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof), thereby proliferating stem cells in the stem cell population and resulting in an expanded population of stem cells; and exposing the expanded population of stem cells to a GSK3-beta inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and/or a Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof), thereby facilitating generation of inner ear hair cells from the expanded population of stem cells.

In certain embodiments, the disclosure provides methods for preventing and treating auditory dysfunction. For example, in certain embodiments, the disclosure provides methods for preventing or treating auditory impairments or restoring and improving hearing in a subject comprising administering to said subject an effective amount of a composition herein describe.

The methods and compositions of the present disclosure allow greater and thus more effective dosing with these ototoxicity-inducing pharmaceutical drugs, while concomitantly preventing or reducing ototoxic effects caused by these drugs. The methods of the present disclosure provide a safe, effective, and prolonged means for prophylactic or curative treatment of hearing impairments related to inner ear tissue damage, loss, or degeneration, particularly sound or aging-induced, and ototoxin-induced, and particularly involving inner ear hair cells. In certain embodiments, the present disclosure provides compositions and methods that address one or more of these or other goals.

This disclosure relates to compositions useful in methods for inducing, promoting, or enhancing the growth, proliferation, or regeneration of inner ear tissue, for example, inner ear supporting cells and/or inner ear hair cells.

The present disclosure relates to methods and compositions to activate the Wnt pathway or to inhibit GSK3-beta activity.

In another aspect the present disclosure provides to compositions and methods for controlled proliferation of stem cells comprising administering to a cell population an effective amount of a composition provided herein.

In another aspect the present disclosure relates to compositions and methods to prevent, reduce or treat the incidence and/or severity of disorders or diseases associated with absence or lack of certain tissue cells. In one aspect the present disclosure relates to compositions and methods to prevent, reduce or treat the incidence and/or severity of inner ear disorders and hearing impairments involving inner ear tissue, particularly inner ear hair cells, their progenitors, and optionally, the stria vascularis, and associated auditory nerves. Of particular interest are those conditions that lead to permanent hearing loss where reduced number of hair cells may be responsible and/or decreased hair cell function. Also of interest are those arising as an unwanted side-effect of ototoxic therapeutic drugs including cisplatin and its analogs, aminoglycoside antibiotics, salicylate and its analogs, or loop diuretics. In certain embodiments, the present disclosure relates to inducing, promoting, or enhancing the growth, proliferation or regeneration of inner ear tissue, particularly inner ear supporting cells and hair cells.

When cochlea supporting cell populations are treated with the compositions described herein, whether the population is in vivo or in vitro, the treated supporting cells exhibit stem-like behavior in that the treated supporting cells have the capacity to proliferate and differentiate and, more specifically, differentiate into cochlear hair cells. Alternatively, the composition induces and maintains the supporting cells to produce daughter stem cells that can divide for many generations and maintain the ability to have a high proportion of the resulting cells differentiate into hair cells. In certain embodiments, the proliferating stem cells express stem cell markers which may include Lgr5, Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1, Tc11, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1, Smad2, smad2/3, smad4, smad5, and/or smad7.

In some embodiments, the method of the present disclosure may be used to maintain, or even transiently increase stemness (i.e., self-renewal) of a pre-existing supporting cell population prior to significant hair cell formation. In some embodiments, the pre-existing supporting cell population comprises inner pillar cells, outer pillar cells, inner phalangeal cells, Deiter cells, Hensen cells, Boettcher cells, and/or Claudius cells. Morphological analyses with immunostaining (including cell counts) and lineage tracing across a Representative Microscopy Samples may be used to confirm expansion of one or more of these cell-types. In some embodiments, the pre-existing supporting cells comprise Lgr5⁺ cells. Morphological analyses with immunostaining (including cell counts) and qPCR and RNA hybridization may be used to confirm Lgr5 upregulation amongst the cell population.

Advantageously, the methods of the present disclosure achieve these goals without the use of genetic manipulation. Germ-line manipulation used in many academic studies is not a therapeutically desirable approach to treating hearing loss. In general, the therapy involves the administration of a small molecule, peptide, antibody, or other non-nucleic acid molecule or nucleic acid delivery vector unaccompanied by gene therapy. In certain embodiments, the therapy involves the administration of a small organic molecule. Alternatively, hearing protection or restoration is achieved through the use of a (non-genetic) therapeutic that is injected in the middle ear and diffuses into the cochlea.

The cochlea relies heavily on all present cell types, and the organization of these cells is important to their function. As supporting cells play an important role in neurotransmitter cycling and cochlear mechanics. Thus, maintaining a rosette patterning within the organ of Corti may be important for function. Cochlear mechanics of the basilar membrane activate hair cell transduction. Due to the high sensitivity of cochlear mechanics, it is also desirable to avoid masses of cells. In all, maintaining proper distribution and relation of hair cells and supporting cells along the basilar membrane, even after proliferation, is likely a desired feature for hearing as supporting cell function and proper mechanics is necessary for normal hearing.

In one embodiment of the present disclosure, the cell density of hair cells in a cochlear cell population is expanded in a manner that maintains, or even establishes, the rosette pattern characteristic of cochlear epithelia.

In accordance with one aspect of the present disclosure, the cell density of hair cells may be increased in a population of cochlear cells comprising both hair cells and supporting cells. The cochlear cell population may be an in vivo population (i.e., comprised by the cochlear epithelium of a subject) or the cochlear cell population may be an in vitro (ex vivo) population. If the population is an in vitro population, the increase in cell density may be determined by reference to a Representative Microscopy Sample of the population taken prior and subsequent to any treatment. If the population is an in vivo population, the increase in cell density may be determined indirectly by determining an effect upon the hearing of the subject with an increase in hair cell density correlating to an improvement in hearing.

In one embodiment, supporting cells placed in a Stem Cell Proliferation Assay in the absence of neuronal cells form ribbon synapses.

In a native cochlea, patterning of hair cells and supporting cells occurs in a manner parallel to the basilar membrane. In one embodiment of the present disclosure, the proliferation of supporting cells in a cochlear cell population is expanded in a manner that the basilar membrane characteristic of cochlear epithelia.

In one embodiment, the number of supporting cells in an initial cochlear cell population is selectively expanded by treating the initial cochlear cell population with a composition comprising the composition comprising the composition comprising the salt of Formula I to form an intermediate cochlear cell population and wherein the ratio of supporting cells to hair cells in the intermediate cochlear cell population exceeds the ratio of supporting cells to hair cells in the initial cochlear cell population. The expanded cochlear cell population may be, for example, an in vivo population, an in vitro population or even an in vitro explant. In one such embodiment, the ratio of supporting cells to hair cells in the intermediate cochlear cell population exceeds the ratio of supporting cells to hair cells in the initial cochlear cell population. For example, in one such embodiment the ratio of supporting cells to hair cells in the intermediate cochlear cell population exceeds the ratio of supporting cells to hair cells in the initial cochlear cell population by a factor of 1.1. By way of further example, in one such embodiment the ratio of supporting cells to hair cells in the intermediate cochlear cell population exceeds the ratio of supporting cells to hair cells in the initial cochlear cell population by a factor of 1.5. By way of further example, in one such embodiment the ratio of supporting cells to hair cells in the intermediate cochlear cell population exceeds the ratio of supporting cells to hair cells in the initial cochlear cell population by a factor of 2. By way of further example, in one such embodiment the ratio of supporting cells to hair cells in the intermediate cochlear cell population exceeds the ratio of supporting cells to hair cells in the initial cochlear cell population by a factor of 3. In each of the foregoing embodiments, the capacity of a composition of the present disclosure to expand a cochlear cell population as described in this paragraph may be determined by means of a Stem Cell Proliferation Assay.

In one embodiment, the number of stem cells in a cochlear cell population is expanded to form an intermediate cochlear cell population by treating a cochlear cell population with a composition comprising the composition comprising the composition comprising the salt of Formula I wherein the cell density of stem cells in the intermediate cochlear cell population exceeds the cell density of stem cells in the initial cochlear cell population. The treated cochlear cell population may be, for example, an in vivo population, an in vitro population or even an in vitro explant. In one such embodiment, the cell density of stem cells in the treated cochlear cell population exceeds the cell density of stem cells in the initial cochlear cell population by a factor of at least 1.1. For example, in one such embodiment the cell density of stem cells in the treated cochlear cell population exceeds the cell density of stem cells in the initial cochlear cell population by a factor of at least 1.25. For example, in one such embodiment the cell density of stem cells in the treated cochlear cell population exceeds the cell density of stem cells in the initial cochlear cell population by a factor of at least 1.5. By way of further example, in one such embodiment the cell density of stem cells in the treated cochlear cell population exceeds the cell density of stem cells in the initial cochlear cell population by a factor of at least 2. By way of further example, in one such embodiment the cell density of stem cells in the treated cochlear cell population exceeds the cell density of stem cells in the initial cochlear cell population by a factor of at least 3. In vitro cochlear cell populations may expand significantly more than in vivo populations; for example, in certain embodiments the cell density of stem cells in an expanded in vitro population of stem cells may be at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2,000 or even 3,000 times greater than the cell density of the stem cells in the initial cochlear cell population. In each of the foregoing embodiments, the capacity of a composition of the present disclosure to expand a cochlear cell population as described in this paragraph may be determined by means of a Stem Cell Proliferation Assay.

In accordance with one aspect of the present disclosure, a cochlea supporting cell population is treated with a composition comprising the composition comprising the salt of Formula I to increase the Lgr5 activity of the population. For example, in one embodiment the composition comprising the salt of Formula I has the capacity to increase and maintain the Lgr5 activity of an in vitro population of cochlea supporting cells by factor of at least 1.2. By way of further example, in one such embodiment the composition has the capacity to increase the Lgr5 activity of an in vitro population of cochlea supporting cells by factor of 1.5. By way of further example, in one such embodiment the composition has the capacity to increase the Lgr5 activity of an in vitro population of cochlea supporting cells by factor of 2, 3, 5 10, 100, 500, 1,000, 2,000 or even 3,000. Increases in Lgr5 activity may also be observed for in vivo populations but the observed increase may be somewhat more modest. For example, in one embodiment the composition has the capacity to increase the Lgr5 activity of an in vivo population of cochlea supporting cells by at least 5%. By way of further example, in one such embodiment the composition has the capacity to increase the Lgr5 activity of an in vivo population of cochlea supporting cells by at least 10%. By way of further example, in one such embodiment the composition has the capacity to increase the Lgr5 activity of an in vivo population of cochlea supporting cells by at least 20%. By way of further example, in one such embodiment the composition has the capacity to increase the Lgr5 activity of an in vivo population of cochlea supporting cells by at least 30%. In each of the foregoing embodiments, the capacity of the composition for such an increase in Lgr5 activity may be demonstrated, for example, in an In vitro Lgr5⁺ Activity Assay and in an in vivo population may be demonstrated, for example, in an In vivo Lgr5⁺ Activity Assay, as measured by isolating the organ and performing morphological analyses using immunostaining, endogenous fluorescent protein expression of Lgr5 (e.g., Lgr5, Sox2), and qPCR for Lgr5.

In addition to increasing the Lgr5 activity of the population, the number of Lgr5⁺ supporting cells in a cochlea cell population may be increased by treating a cochlea cell population containing Lgr5⁺ supporting cells (whether in vivo or in vitro) with a composition comprising the composition comprising the salt of Formula I. In general, the cell density of the stem/progenitor supporting cells may expand relative to the initial cell population via one or more of several mechanisms. For example, in one such embodiment, newly generated Lgr5⁺ supporting cells may be generated that have increased stem cell propensity (i.e., greater capacity to differentiate into hair cell). By way of further example, in one such embodiment no daughter Lgr5⁺ cells are generated by cell division, but pre-existing Lgr5⁺ supporting cells are induced to differentiate into hair cells. By way of further example, in one such embodiment no daughter cells are generated by cell division, but Lgr5⁻ supporting cells are activated to a greater level of Lgr5 activity and the activated supporting cells are then able to differentiate into hair cells. Regardless of the mechanism, in one embodiment the composition of the present disclosure has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vitro isolated cell population of cochlea supporting cells by factor of at least 5. By way of further example, in one such embodiment the composition has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vitro population of cochlea supporting cells by factor of at least 10. By way of further example, in one such embodiment the composition has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vitro population of cochlea supporting cells by factor of at least 100, at least 500, at least 1,000 or even at least 2,000. Increases in the cell density of Lgr5⁺ supporting cells may also be observed for in vivo populations but the observed increase may be somewhat more modest. For example, in one embodiment the composition has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vivo population of cochlea supporting cells by at least 5%. By way of further example, in one such embodiment the composition has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vivo population of cochlea supporting cells by at least 10%. By way of further example, in one such embodiment the composition has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vivo population of cochlea supporting cells by at least 20%. By way of further example, in one such embodiment the composition has the capacity to increase the cell density of Lgr5⁺ supporting cells in an in vivo population of cochlea supporting cells by at least 30%. The capacity of the composition for such an increase in Lgr5⁺ supporting cells in an in vitro population may be demonstrated, for example, in a Stem Cell Proliferation Assay or in an appropriate in vivo assay. In one embodiment, a composition of the present disclosure has the capacity to increase the number of Lgr5⁺ cells in the cochlea by inducing expression of Lgr5 in cells with absent or low detection levels of the protein, while maintaining Native Morphology. In one embodiment, a composition of the present disclosure has the capacity to increase the number of Lgr5⁺ cells in the cochlea by inducing expression of Lgr5 in cells with absent or low detection levels of the protein, while maintaining Native Morphology and without producing Cell Aggregates.

In addition to increasing the cell density of Lgr5⁺ supporting cells, in one embodiment the method of the present disclosure has the capacity to increase the ratio of Lgr5⁺ cells to hair cells in a cochlear cell population. In one embodiment, the number of Lgr5⁺ supporting cells in an initial cochlear cell population is selectively expanded by treating the initial cochlear cell population with a composition of the present disclosure to form an expanded cell population and wherein the number of Lgr5⁺ supporting cells in the expanded cochlear cell population at least equals the number of hair cells. The expanded cochlear cell population may be, for example, an in vivo population, an in vitro population or even an in vitro explant. In one such embodiment, the ratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlear cell population is at least 1:1. For example, in one such embodiment the ratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlear cell population is at least 1.5:1. By way of further example, in one such embodiment the ratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlear cell population is at least 2:1. By way of further example, in one such embodiment the ratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlear cell population is at least 3:1. By way of further example, in one such embodiment the ratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlear cell population is at least 4:1. By way of further example, in one such embodiment the ratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlear cell population is at least 5:1. In each of the foregoing embodiments, the capacity of the composition of the present disclosure to expand a cochlear cell population as described in this paragraph may be determined by means of a Stem Cell Proliferation Assay.

In certain embodiments, the method increases the fraction of the Lgr5⁺ cells to total cells on the sensory epithelium by at least 10%, 20%, 50%, 100%, 250% 500%, 1,000% or 5000%.

In certain embodiments, the method increases the Lgr5⁺ cells until they become at least 10, 20, 30, 50, 70, or 85% of the cells on the sensory epithelium, e.g., the Organ of Corti.

In general, excessive proliferation of supporting cells in the cochlea is avoided. In one embodiment, the method of the present disclosure has the capacity to expand a cochlear cell population without creating a protrusion of new cells beyond the native surface of the cochlea, e.g., a Cell Aggregate. In some embodiments, 30 days after placing a composition comprising the composition comprising the salt of Formula I on the round or oval window membrane, the cochlear tissue has Native Morphology. In some embodiments, 30 days after placing the composition on the round or oval window membrane, the cochlear tissue has Native Morphology and lacks Cell Aggregates. In some embodiments, 30 days after placing the composition on the round or oval window membrane, the cochlear tissue has Native Morphology and at least 10, 20, 30, 50, 75, 90, 95, 98, or even at least 99% of the Lgr5⁺ cells in the Organ of Corti are not part of Cell Aggregates.

In addition to expanding supporting cell populations, generally, and Lgr5⁺ supporting cells, specifically, as described above, the method of the present disclosure has the capacity to maintain, in the daughter cells, the capacity to differentiate into hair cells. In in vivo populations, the maintenance of this capacity may be indirectly observed by an improvement in a subject's hearing. In in vitro populations, the maintenance of this capacity may be directly observed by an increase in the number of hair cells relative to a starting population or indirectly by measuring LGR5 activity, SOX2 activity or one or more of the other stem cell markers identified elsewhere herein.

In one embodiment, the capacity of the method to increase the stemness of a population of cochlear supporting cells, in general, or a population of Lgr5⁺ supporting cells, in particular, may be correlated with an increase of Lgr5 activity of an in vitro population of isolated Lgr5⁺ cells as determined by an Lgr5 Activity Assay. As previously noted, in one such embodiment, the composition has the capacity to increase the Lgr5 activity of stem cells in the intermediate cell population by a factor of 5 on average relative to the Lgr5 activity of the cells in the initial cell population. By way of further example, in one such embodiment the method has the capacity to increase the Lgr5 activity of the stem cells genes in the intermediate cell population by a factor of 10 relative to the Lgr5 activity of the cells in the initial cell population. By way of further example, in one such embodiment the method has the capacity to increase the Lgr5 activity of the stem cells in the intermediate cell population by a factor of 100 relative to the Lgr5 activity of the cells in the initial cell population. By way of further example, in one such embodiment the method has the capacity to increase the Lgr5 activity of the stem cells in the intermediate cell population by a factor of 1000 relative to the Lgr5 activity of the cells in the initial cell population. In each of the foregoing embodiments, the increase in the activity of stem cells in the cell population may be determined in vitro by immunostaining or endogenous fluorescent protein expression for target genes and analysis of their relative intensities via imaging analysis or flowcytometry, or using qPCR for target stem cell genes. The identity of the resulting stem cell population may optionally be further determined by stem cell assays including stem cell marker expression assay, colony forming assay, self-renewal assay and differentiation assay as defined in Stem cell assay.

In some embodiments, the method applied to an adult mammal produces a population of adult mammalian Lgr5⁺ cells that are in S-phase.

In one embodiment, after applying the composition comprising the composition comprising the salt of Formula I to the round or oval window of a mouse, the in vivo Lgr5⁺ Activity of a cell population in the Organ of Corti increases 1.3×, 1.5×, up to 20× over baseline for a population that has not been exposed to the composition. In some embodiments, applying the composition to the round or oval window of a mouse increases the average In vivo Lgr5⁺ Activity for cells in the Organ of Corti is increased 1.3×, 1.5×, up to 20× over baseline for a population that has not been exposed to the composition.

In certain embodiments, the method increases the Lgr5⁺ cells until they become at least 10%, 7.5%, 10%, up to 100% of the supporting cell population by number.

In certain embodiments, the composition has the capacity to increase the percentage of Lgr5⁺ cell in a cochlea by 5%, 10%, 25%, 50%, or 80%.

In certain embodiments, the stem cell population is of an in vivo subject, and the method is a treatment for hearing loss and/or vestibular dysfunction (e.g., wherein the generation of inner ear hair cells from the expanded population of stem cells results in partial or full recovery of hearing loss and/or improved vestibular function). In certain embodiments, the stem cell population is of an in vivo subject, and the method further comprises delivering a drug to the subject (e.g., for treatment of a disease and/or disorder unrelated to hearing loss and/or vestibular dysfunction) at a higher concentration than a known safe maximum dosage of the drug for the subject (e.g., the known safe maximum dosage if delivered in the absence of the generation of inner ear hair cells resulting from the method) (e.g., due to a reduction or elimination of a dose-limiting ototoxicity of the drug).

In certain embodiments, the method further comprises performing high throughput screening using the generated inner ear hair cells. In certain embodiments, the method comprises using the generated inner ear hair cells to screen molecules for toxicity against inner ear hair cells. In certain embodiments, the method comprises using the generated inner ear hair cells to screen molecules for ability to improve survival of inner ear hair cells (e.g., inner ear hair cells exposed to said molecules).

In another aspect, the disclosure is directed to a method of producing an expanded population of stem cells, the method comprising: administering or causing to be administered to a stem cell population (e.g., of an in vitro, ex vivo, or in vivo sample/subject) a composition comprising the composition comprising the salt of Formula I.

In certain embodiments, the administering step is carried out by performing one or more injections into the ear (e.g., transtympanically into the middle ear and/or inner ear).

In certain embodiments, the administering step comprises administering the GSK3-beta inhibitor and/or Wnt agonist in a sustained manner.

In certain embodiments, the stem cells are inner ear stem cells and/or supporting cells.

In certain embodiments, the method further comprises performing high throughput screening using the generated expanded population of stem cells. In certain embodiments, the method further comprises using the generated stem cells to screen molecules for toxicity against stem cells and/or their progeny. In certain embodiments, the method comprises using the generated stem cells to screen molecules for ability to improve survival of stem cells and/or their progeny.

In another aspect, the disclosure is directed to a method of treating a subject who has, or is at risk of developing, hearing loss and/or vestibular dysfunction, the method comprising: identifying a subject who has experienced, or is at risk for developing, hearing loss and/or vestibular dysfunction, administering or causing to be administered a composition comprising any of the compositions or compounds provided herein.

In certain embodiments, the stem cell population comprises Lgr5⁺ cells. In certain embodiments, the stem cell population comprises post-natal cells. In certain embodiments, the stem cell population comprises epithelial stem cells. In certain embodiments, stem cells include progenitor cells.

In certain embodiments, the step of administering is carried out by performing one or more injections into the ear (e.g., transtympanically into the middle ear and/or inner ear).

In another aspect, the disclosure is directed to a method of generating inner ear hair cells, the method comprising: proliferating stem cells in an initial stem cell population (e.g., of an in vitro, ex vivo, or in vivo sample/subject), resulting in an expanded population of stem cells (e.g., such that the expanded population is a factor of at least 1.25, 1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cell population); and facilitating generation of inner ear hair cells from the expanded population of stem cells.

In another aspect, the disclosure is directed to a method of generating inner ear hair cells, the method comprising administering a composition comprising the composition comprising the salt of Formula I (e.g., in a pharmaceutically acceptable form (e.g., salt)) to a cell population in an inner ear of a subject, thereby facilitating generation of inner ear hair cells.

In another aspect, the disclosure is directed to a method of generating inner ear hair cells, the method comprising: proliferating post-natal LGR5+ cells in an initial population (e.g., of an in vitro, ex vivo, or in vivo sample/subject), resulting in an expanded population of LGR5+ cells (e.g., such that the expanded population is a factor of at least 1.25, 1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cell population), said expanded population of LGR5+ cells resulting in generation of inner ear hair cells. In certain embodiments, stem cells include progenitor cells.

In another aspect, the disclosure is directed to a method of treating a disease or disorder, the method comprising: proliferating post-natal Lgr5⁺ epithelial cells in an initial population of a subject (in vivo), resulting in an expanded population of Lgr5+ epithelial cells (e.g., such that the expanded population is a factor of at least 1.25, 1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial post-natal Lgr5⁺ epithelial cell population).

In some embodiments, Lgr5⁺ cells are differentiated into hair cells.

In some embodiments of the compositions described herein, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 0.01 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 0.1 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 1 uM to about 100 mM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 10 uM to about 10 mM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 1 uM to about 10 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 10 uM to about 100 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 100 uM to about 1000 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 1 mM to about 10 mM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor or GSK-3 beta inhibitor is used at a concentration of about 10 mM to about 100 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 1 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 10 uM to about 100 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 100 uM to about 100 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 1 mM to about 10 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is LY2090314 used at a concentration of about 0.01 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 0.1 uM to about 10 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 1 uM to about 1 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 1 uM to about 100 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 10 uM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 0.1 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 1 uM to about 100 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 10 uM to about 10 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 100 uM to about 10 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 100 uM to about 1 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 0.1 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 1 uM to about 1,000 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 10 uM to about 100 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 100 uM to about 10 mM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 1 mM to about 10 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the HDAC inhibitor is used at a concentration of about 0.01 uM to about 100,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1 uM to about 10,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 10 uM to about 10000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 100 uM to about 1000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1 uM to about 10 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 10 uM to about 100 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 100 uM to about 1000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1000 uM to about 10 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 10 mM to about 100 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 100 mM to about 1000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1000 mM to about 10,000 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the HDAC inhibitor is Valproic Acid used at a concentration of about 10 uM to about 100,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 1 mM to about 10,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 10 mM to about 10,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 100 mM to about 10,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 200 mM to about 2,000 mM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 1,000 mM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 0.01 to about 1,000,000-fold the activity in Effective Stemness Driver Concentration and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 0.1 to about 100,000-fold the Effective Stemness Driver Concentration and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 1 to about 10,000-fold the Effective Stemness Driver Concentration and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 100 to about 5000-fold the Effective Stemness Driver Concentration and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 50 to about 2000-fold the Effective Stemness Driver Concentration and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 100 to about 1000-fold the Effective Stemness Driver Concentration and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitor, or GSK-3 beta inhibitor is used at a concentration ratio of about 1,000-fold the Effective Stemness Driver Concentration and optionally in combination with other agents.

In some embodiments of the compositions described herein, the HDAC inhibitor is used at a concentration ratio of about 0.1 to about 1,000,000-fold the Effective Concentration and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration ratio of about 1 to about 100,000-fold the Effective Concentration and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration ratio of about 10 to about 10,000-fold the Effective Concentration and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration ratio of about 100 to about 1000-fold the Effective Concentration and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration ratio of about 1,000-fold the Effective Concentration and optionally in combination with other agents.

In some embodiments of the compositions described herein, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 0.01 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 0.1 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 1 nM to about 100 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 10 nM to about 10 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 1 nM to about 10 nM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 10 nM to about 100 nM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 100 nM to about 1000 nM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 1 uM to about 10 uM and optionally in combination with other agents. In other embodiments, the Wnt agonist, GSK-3 alpha inhibitors or GSK-3 beta inhibitor is used at a concentration of about 10 uM to about 100 uM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 1 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 10 nM to about 100 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 100 nM to about 100 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is CHIR99021 used at a concentration of about 1 uM to about 10 uM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is LY2090314 used at a concentration of about 0.01 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 0.1 nM to about 10 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 1 nM to about 1 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 1 nM to about 100 nM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is LY2090314 used at a concentration of about 10 nM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 0.1 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 1 nM to about 100 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 10 nM to about 10 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is GSK inhibitor XXII used at a concentration of about 100 nM to about 1 uM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 1 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 10 nM to about 1,000 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 100 nM to about 100 uM and optionally in combination with other agents. In other embodiments, the GSK-3 inhibitor is AZD 1080 used at a concentration of about 1 uM to about 10 uM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the HDAC inhibitor is used at a concentration of about 0.01 nM to about 100,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1 nM to about 10,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 10 nM to about 10,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 100 nM to about 1000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1 nM to about 10 nM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 10 nM to about 100 nM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 100 nM to about 1000 nM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1 uM to about 10 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 10 uM to about 100 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 100 uM to about 1000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is used at a concentration of about 1,000 uM to about 10,000 uM and optionally in combination with other agents.

In some embodiments of the compositions described herein, the HDAC inhibitor is Valproic Acid used at a concentration of about 10 nM to about 100,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 1 uM to about 10,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 10 uM to about 10,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 100 uM to about 10,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 200 uM to about 2,000 uM and optionally in combination with other agents. In other embodiments, the HDAC inhibitor is Valproic Acid used at a concentration of about 1,000 uM and optionally in combination with other agents.

Administration

The membrane of the round or oval window is the biological barrier to the inner ear space and represents the major obstacle for the local treatment of hearing impairment. The administered drug must overcome this membrane to reach the inner ear space. The drug can operatively (e.g., injection through the tympanic membrane) be placed locally to the round or oval window membrane and can then penetrate through the round or oval window membrane. Substances that penetrate the round or oval window typically distribute in the perilymph and thus reach the hair cells and supporting cells.

In certain embodiments, pharmaceutical compositions are adapted to administer the drug locally to the round or oval window membrane. The pharmaceutical compositions may also contain a membrane penetration enhancer, which supports the passage of the agents mentioned herein through the round or oval window membrane. Accordingly, liquid, gel or foam compositions may be used. It is also possible to apply the active ingredient orally or to employ a combination of delivery approaches.

Intratympanic (IT) delivery of drugs to the ear is increasingly used for both clinical and research purposes. Some groups have applied drugs in a sustained manner using microcatheters and microwicks, while the majority have applied them as single or as repeated IT injections (up to 8 injections over periods of up to 2 weeks)8.

Intratympanically applied drugs are thought to enter the fluids of the inner ear primarily by crossing the round or oval window (RW) membrane. Calculations show that a major factor controlling both the amount of drug entering the ear and the distribution of drug along the length of the ear is the duration the drug remains in the middle ear space. Single, ‘one-shot’ applications or applications of aqueous solutions for few hours' duration result in steep drug gradients for the applied substance along the length of the cochlea and rapidly declining concentration in the basal turn of the cochlea as the drug subsequently becomes distributed throughout the ear.

Other injection approaches include by osmotic pump, or, by combination with implanted biomaterial, by injection or infusion. Biomaterials that can aid in controlling release kinetics and distribution of drug include hydrogel materials, degradable materials. One class of materials that is used includes in situ gelling materials. All potential materials and methodologies mentioned in these references are included herein by reference (Almeida H, Amaral M H, Lobao P, Lobo J M. In situ gelling systems: a strategy to improve the bioavailability of ophthalmic pharmaceutical compositions. Drug Discovery Today 2014; 19:400-12; Wise A K, Gillespie L N. Drug delivery to the inner ear. Journal of Neural Engineering 2012; 9:065002; Surovtseva E V, Johnston A H, Zhang W, et al. Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear. International Journal of Pharmaceutics 2012; 424:121-7; Roy S, Glueckert R, Johnston A H, et al. Strategies for drug delivery to the human inner ear by multifunctional nanoparticles. Nanomedicine 2012; 7:55-63; Rivera T, Sanz L, Camarero G, Varela-Nieto I. Drug delivery to the inner ear: strategies and their therapeutic implications for sensorineural hearing loss. Current Drug Delivery 2012; 9:231-42; Pararas E E, Borkholder D A, Borenstein J T. Microsystems technologies for drug delivery to the inner ear. Advanced drug delivery reviews 2012; 64:1650-60; Li M L, Lee L C, Cheng Y R, et al. A novel aerosol-mediated drug delivery system for inner ear therapy: intratympanic aerosol methylprednisolone can attenuate acoustic trauma. IEEE Transactions on Biomedical Engineering 2013; 60:2450-60; Lajud S A, Han Z, Chi F L, et al. A regulated delivery system for inner ear drug application. Journal of controlled release: official journal of the Controlled Release Society 2013; 166:268-76; Kim D K, Park S N, Park K H, et al. Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles. Drug delivery 2014; Kanzaki S, Fujioka M, Yasuda A, et al., PloS ONE 2012; 7:e48480; Engleder E, Honeder C, Klobasa J, Wirth M, Arnoldner C, Gabor F. Preclinical evaluation of thermoreversible triamcinolone acetonide hydrogels for drug delivery to the inner ear. International Journal of Pharmaceutics 2014; 471:297-302; Bohl A, Rohm H W, Ceschi P, et al. Development of a specially tailored local drug delivery system for the prevention of fibrosis after insertion of cochlear implants into the inner ear. Journal of Materials Science: Materials in Medicine 2012; 23:2151-62; Hoskison E, Daniel M, Al-Zahid S, Shakesheff K M, Bayston R, Birchall J P. Drug delivery to the ear. Therapeutic Delivery 2013; 4:115-24; Staecker H, Rodgers B., Expert Opin Drug Deliv 2013; 10:639-50; Pritz C O, Dudas J, Rask-Andersen H, Schrott-Fischer A, Glueckert R. Nanomedicine strategies for drug delivery to the ear. Nanomedicine 2013; 8:1155-72), which are included herein by reference in their entirety. Other materials include collagen or other natural materials including fibrin, gelatin, and decellularized tissues. Gelfoam may also be suitable.

Delivery may also be enhanced via alternate means including but not limited to agents added to the delivered composition such as penetration enhancers, or could be through devices via ultrasound, electroporation, or high speed jet.

Methods described herein can also be used for inner ear cell types that may be produced using a variety of methods know to those skilled in the art including those cell types described in PCT Application No. WO2012103012 A1.

With regard to human and veterinary treatment, the amount of a particular agent(s) that is administered may be dependent on a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific agent(s) employed; the duration of the treatment; drugs used in combination or coincidental with the specific agent(s) employed; the judgment of the prescribing physician or veterinarian; and like factors known in the medical and veterinary arts.

The agents described herein may be administered in a therapeutically effective amount to a subject in need of treatment. Administration of compositions described herein can be via any of suitable route of administration, particularly by intratympanically. Other routes include ingestion, or alternatively parenterally, for example intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly, intranasally, subcutaneously, sublingually, transdermally, or by inhalation or insufflations, or topical by ear instillation for absorption through the skin of the ear canal and membranes of the eardrum. Such administration may be as a single or multiple oral doses, defined number of ear drops, or a bolus injection, multiple injections, or as a short- or long-duration infusion. Implantable devices (e.g., implantable infusion pumps) may also be employed for the periodic parenteral delivery over time of equivalent or varying dosages of the particular composition. For such parenteral administration, the composition s are formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances such as salts, sugars (particularly glucose or mannitol), to make the solution isotonic with blood, buffering agents such as acetic, citric, and/or phosphoric acids and their sodium salts, and preservatives. The preparation of suitable and sterile parenteral compositions is described in detail in the section entitled “Compositions” above.

Compositions described herein can be administered by a number of methods sufficient to deliver the composition to the inner ear. Delivering a composition to the inner ear includes administering the composition to the middle ear, such that the composition may diffuse across the round or oval window to the inner ear and administering a composition to the inner ear by direct injection through the round or oval window membrane. Such methods include, but are not limited to auricular administration, by transtympanic wicks or catheters, or parenteral administration, for example, by intraauricular, transtympanic, or intracochlear injection.

In particular embodiments, the compositions and compositions of the disclosure are locally administered, meaning that they are not administered systemically.

In one embodiment, a syringe and needle apparatus is used to administer compositions to a subject using auricular administration. A suitably sized needle is used to pierce the tympanic membrane and a wick or catheter comprising the composition is inserted through the pierced tympanic membrane and into the middle ear of the subject. The device may be inserted such that it is in contact with the round or oval window or immediately adjacent to the round or oval window. Exemplary devices used for auricular administration include, but are not limited to, transtympanic wicks, transtympanic catheters, round or oval window microcatheters (small catheters that deliver medicine to the round or oval window), and Silverstein Microwicks™ (small tube with a “wick” through the tube to the round or oval window, allowing regulation by subject or medical professional).

In another embodiment, a syringe and needle apparatus is used to administer compositions to a subject using transtympanic injection, injection behind the tympanic membrane into the middle and/or inner ear. The composition may be administered directly onto the round or oval window membrane via transtympanic injection or may be administered directly to the cochlea via intracochlear injection or directly to the vestibular organs via intravestibular injection.

In some embodiments, the delivery device is an apparatus designed for administration of compositions to the middle and/or inner ear. By way of example only: GYRUS Medical Gmbh offers micro-otoscopes for visualization of and drug delivery to the round or oval window niche; Arenberg has described a medical treatment device to deliver fluids to inner ear structures in U.S. Pat. Nos. 5,421,818; 5,474,529; and 5,476,446, each of which is incorporated by reference herein for such disclosure. U.S. patent application Ser. No. 08/874,208, which is incorporated herein by reference for such disclosure, describes a surgical method for implanting a fluid transfer conduit to deliver compositions to the inner ear. U.S. Patent Application Publication 2007/0167918, which is incorporated herein by reference for such disclosure, further describes a combined otic aspirator and medication dispenser for transtympanic fluid sampling and medicament application.

In some embodiments, a composition disclosed herein is administered to a subject in need thereof once. In some embodiments, a composition disclosed herein is administered to a subject in need thereof more than once. In some embodiments, a first administration of a composition disclosed herein is followed by a second, third, fourth, or fifth administration of a composition disclosed herein.

The number of times a composition is administered to a subject in need thereof depends on the discretion of a medical professional, the disorder, the severity of the disorder, and the subject's response to the composition. In some embodiments, a composition disclosed herein is administered once to a subject in need thereof with a mild acute condition. In some embodiments, a composition disclosed herein is administered more than once to a subject in need thereof with a moderate or severe acute condition. In the case wherein the subject's condition does not improve, upon the doctor's discretion the composition may be administered chronically, that is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.

In the case wherein the subject's status does improve, upon the doctor's discretion the composition may administered continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dose reduction during a drug holiday may be from 10%-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once the subject's hearing and/or balance has improved, a maintenance dose can be administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, is optionally reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, subjects require intermittent treatment on a long-term basis upon any recurrence of symptoms

EXAMPLES Example 1: Effects of Poloxamer and Valproate on CHIR99021 Dihydrochloride (diHCl)

A 23.75% (w/v) poloxamer solution was prepared in 1×PBS containing 1M (166.2 mg/ml) Valproic acid sodium salt, 5% (v/v) DMSO, and 2.77 mM (1.39 mg/ml) CHIR99021 freebase. The poloxamer was fully soluble in the solutions at this concentration and the solution retained fluidity at 4° C. as judged by passage through a micropipette with a 100 uM opening. The solution also retained poloxamer polymerization capacity at 37° C. as judged by gelling of 10 ul of solution pipetted onto coverglass warmed to 37° C. in less than 20 seconds. The final pH of this solution is around 9.

Example 2: Effects on Solubility at Various Concentrations of Poloxamer

P407 solubility was studied at 16.7%, 20%, 23.1%, 25.9%, 28.7%, 33.3%, 37.5%, 41.2%, and 50% w/w in 1M Valproic acid sodium salt and 5% dimethylsulfoxide (DMSO). Valproic acid sodium salt (NaVal) stock solution was prepared by dissolving 16.62 g Valproic acid sodium salt+5.0 g DMSO+78.37 g Water. Poloxamer 407 (P407) was weighed and added to containers containing 10 g each of the 1M Valproic acid sodium salt+5% w/w DMSO solution. The solutions were stored overnight in refrigerator at 0-2° C. and observed for any undissolved poloxamer. Poloxamer levels of up to 28.6% are acceptable when formulated with 1 M valproic acid sodium salt and 5% dimethyl sulfoxide.

Results

Samples at a concentration of 16.7% to 23.1% w/w P407 were clear and fluid in cold conditions while solutions at a concentration of 25.9% to 28.6% w/w P407 were clear but viscous. (FIGS. 1 and 2) Concentrations above 33.3% w/w P407 remained undissolved and formed a thick swollen mass. The results demonstrated that poloxamer levels of up to 28.6% are acceptable when formulated with 1 M valproic acid sodium salt and 5% dimethyl sulfoxide. Table 1 below shows the results and observations of the samples at various concentrations of P407.

TABLE 1 % P407 18+ Hours 40+ Hours Composition (w/w) Observations Observations 2 g P407; 10 g 1M NaVal and 16.70% Clear, dissolved solution Clear, dissolved solution 5% DMSO Stock 4 g P407; 10 g 1M NaVal and 28.60% Almost clear, small Clear, dissolved, viscous 5% DMSO Stock white globs gel 5 g P407; 10 g 1M NaVal and 33.30% Slightly swollen white Swollen white glob 5% DMSO Stock glob 6 g P407; 10 g 1M NaVal and 37.50% Swollen white glob Swollen white glob 5% DMSO Stock 7 g P407; 10 g 1M NaVal and 41.20% Swollen white glob Swollen white glob 5% DMSO Stock 10 g P407; 10 g 1M NaVal and   50% Extremely swollen white Extremely swollen white 5% DMSO Stock glob glob 2.5 g P407; 10 g 1M NaVal   20% n/a Clear, dissolved solution and 5% DMSO Stock 3 g P407; 10 g 1M NaVal and 23.10% n/a Clear, dissolved solution 5% DMSO Stock 3.5 g P407; 10 g 1M NaVal 25.90% n/a Clear, dissolved, slightly and 5% DMSO Stock viscous solution

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims 

1.-133.
 134. A poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising: a) a 2-pyrimidinylaminoethylamino-2-pyridine compound; b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and c) a carboxylic acid containing compound; wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.
 135. (canceled)
 136. The composition of claim 134, wherein the 2-pyrimidinylaminoethylamino-2-pyridine containing compound is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile which has the Formula I and pharmaceutically acceptable salts thereof:


137. The composition of claim 134, wherein the Poloxamer 407 is in a concentration between 21 wt % and 40 wt % relative to the composition.
 138. (canceled)
 139. The composition of claim 134, wherein the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition.
 140. The composition of claim 134, wherein the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition.
 141. (canceled)
 142. The composition of claim 134, wherein the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C.
 143. (canceled)
 144. The composition of claim 134, wherein the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. 145.-152. (canceled)
 153. The composition of claim 134, wherein the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H. 154.-157. (canceled)
 158. The composition of claim 134, wherein the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H. 159.-161. (canceled)
 162. The composition of claim 134, wherein the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid. 163.-172. (canceled)
 173. The composition of claim 134, wherein the composition comprises about 8 wt % carboxylic acid. 174.-219. (canceled)
 220. A poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising: a) a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound; b) Poloxamer 407 at greater than or equal to 21 wt % of the pharmaceutical composition; and c) a carboxylic acid containing compound; wherein the pharmaceutical composition has a gelation temperature of greater than 10° C.
 221. The composition of claim 220, wherein the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is a 3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione containing compound.
 222. The composition of claim 220, wherein the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is a 3-(imidazo[1,2-a]pyridin-3-yl)-4-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione containing compound.
 223. The composition of claim 220, wherein 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is 3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione which has the Formula III and pharmaceutically acceptable salts thereof:


224. The composition of claim 220, wherein the Poloxamer 407 is in a concentration between 21 wt % and 40 wt % relative to the composition. 225.-228. (canceled)
 229. The composition of claim 220, wherein the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C. 230.-239. (canceled)
 240. The composition of claim 220, wherein the carboxylic acid containing compound is a substituted or unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H. 241.-244. (canceled)
 245. The composition of claim 220, wherein the carboxylic acid containing compound is an unsubstituted C₅-C₉ straight or branched chain alkyl-CO₂H. 246.-248. (canceled)
 249. The composition of claim 220, wherein the unsubstituted C₉ branched straight chain alkyl-CO₂H carboxylic acid containing compound is Valproic acid. 250.-410. (canceled)
 411. A poloxamer containing pharmaceutical composition having a gelation temperature greater than 10° C., the composition comprising, a) a compound of the Formula I

or a pharmaceutically acceptable salt thereof b) Poloxamer 407 in weight percentage of more than 21% wt; and c) Valproic acid; wherein the amount of Valproic acid sufficient to cause the pharmaceutical composition to have a gelation temperature of greater than 10° C.
 412. The composition of claim 411, wherein the Poloxamer 407 is in a concentration between 21 wt % and 40 wt % relative to the composition.
 413. (canceled)
 414. The composition of claim 411, wherein the Poloxamer 407 is in a concentration between 23 wt % and 29 wt % relative to the composition.
 415. The composition of claim 411, wherein the Poloxamer 407 is in a concentration between 23 wt % and 27 wt % relative to the composition.
 416. The composition of claim 411, wherein the Poloxamer 407 is in a concentration of 25 wt % relative to the composition.
 417. The composition of claim 411, wherein the gelation temperature of the pharmaceutical composition is between about 11° C. and about 32° C.
 418. (canceled)
 419. The composition of claim 411, wherein the gelation temperature of the pharmaceutical composition is between about 20° C. and about 28° C. 420.-423. (canceled)
 424. The composition of claim 411, wherein the gelation temperature of the pharmaceutical composition is about 26° C.
 425. The composition of claim 411, wherein the gelation temperature of the pharmaceutical composition is about 28° C.
 426. (canceled)
 427. (canceled)
 428. The composition of claim 411, wherein the amount of the Valproic acid in the pharmaceutical composition is between least 2 wt % (weight Valproic acid/weight pharmaceutical composition) and 20 wt %.
 429. The composition of claim 411, wherein the composition comprises at least 4 wt % Valproic acid.
 430. The composition of claim 411, wherein the composition comprises at least 8 wt % Valproic acid. 431.-521. (canceled) 